1,3,5-Triazines for treatment of viral diseases

ABSTRACT

The present invention provides compounds and methods for treatment of viral diseases and cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a non-provisional filing of United States ProvisionalApplication No. 60/413,337, filed Sep. 24, 2002, the disclosure of whichis incorporated herein by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] RNA viral diseases are responsible for the vast majority of viralmorbidity and mortality of viral diseases of mankind, including AIDS,hepatitis, rhinovirus infections of the respiratory tract, flu, measles,polio and others. There are a number of chronic persistent diseasescaused by RNA or DNA viruses that replicate through an RNA intermediatewhich are difficult to treat, such as hepatitis B and C, and T-cellhuman leukemia. Many common human diseases are caused by RNA virusesthat are replicated by a viral encoded RNA replicase. Included in thisgroup are influenza (Zurcher, et al., J. Gen. Virol. 77:1745 (1996),dengue fever (Becker, Virus-Genes 9:33 (1994), and rhinovirus infections(Horsnell, et al., J. Gen. Virol., 76:2549 (1995). Important RNA viraldiseases of animals include feline leukemia and immunodeficiency, Visnamaedi of sheep, bovine viral diarrhea, bovine mucosal disease, andbovine leukemia. Although some vaccines are available for DNA viruses,diseases such as hepatitis B are still prevalent. Hepatitis B is causedby a DNA virus that replicates its genome through a RNA intermediate(Summers and Mason, Cell 29:4003 (1982). While an effective vaccineexists as a preventive, there is no efficacious treatment for chronicpersistent HBV infection.

[0003] Chain terminating nucleoside analogs have been used extensivelyfor the treatment of infections by DNA viruses and retroviruses. Theseanalogs are incorporated into DNA by DNA polymerases or reversetranscriptases. Once incorporated, they cannot be further extended andthus terminate DNA synthesis. Unfortunately, there is immediateselective pressure for the development of resistance against such chainterminating analogs that results in development of mutations in theviral polymerase that prevent incorporation of the nucleoside analog.

[0004] An alternative strategy is to utilize mutagenicdeoxyribonucleosides (MDRN) or mutagenic ribonucleosides (MRN) that arepreferentially incorporated into a viral genome. MDRN are incorporatedinto DNA by viral reverse transcriptase or by a DNA polymerase enzyme.MRN are incorporated into viral RNAs by viral RNA replicases. As aresult, the mutations in the viral genome are perpetuated andaccumulated with each viral replication cycle. With each cycle of viralinfection, there ensues a chain like increase in the number of mutationsin the viral genome. Eventually the number of mutations in each viralgenome is so large that no active virally encoded proteins are produced.

[0005] 5-aza-2′-deoxycytidine (5-aza-dC) is an antineoplastic agent thathas been tested in patients with leukemia and is thought to actpredominantly by demethylating DNA. 5-aza-cytidine (5-aza-C) has alsobeen used to treat patients with leukemia. Methylation is thought tosilence tumor growth suppressor and differentiation genes. Interestinglydeamination of 5-aza-dC to 5-aza-2′-deoxyuridine (5-aza-dU) has beenshown to result in loss of antineoplastic activity (see e.g., Momparler,et al., Leukemia. 11:1-6 (1997)). 5-aza-cytidine (5-aza-C) has also beenused to treat patients with leukemia. Both 5-aza-C and 5-aza-dC wereshown to inhibit HIV replication in vitro, although the mechanism ofaction was not determined (see e.g., Bouchard et al, Antimicrob. AgentsChemother. 34: 206-209 (2000)). More recently, 5-aza-C has been shown tobe mutagenic to foot-and-mouth disease virus (see e.g., Sierra et al.,J. Virol. 74(18): 8316-8323 (2000)). Both 5-aza-C and 5-aza-dC areunstable compounds. 5-aza-dC is rapidly degraded upon reconstitution. AtpH 7.0, a 10% degradation occurs at temperatures of 25° C. and 50° C.after 5 and 0.5 hours, respectively (see e.g., Van Groeningen et al.,Cancer Res. 46:4831-4836 (1986)). Thus, therapeutic use of 5-aza-C and5-aza-dC is limited for treatment of both viral diseases and cancer. Thepresent invention solves this and other problems.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides a genus of nucleoside ornucleotide analogues and method of using the analogues as antiviral andanti-cancer chemotherapeutic agents.

[0007] Thus, in a first aspect, there is provided a compound accordingto Formula 1:

[0008] In Formula I, the dashed circle indicates that the ring systemmay include one or more double bonds at any position, such that thevalence of the intra-annular atoms is satisfied. The ring system may bearomatic (e.g., heteroaryl) or non-aromatic. The substituents R², R⁷, R⁸are present or absent as dictated by the application of the laws ofvalency to a selected ring structure.

[0009] The symbol Y represents C, CH or N, and the symbol Z representsC, CH or B. R¹ is a member selected from H, acyl, OR⁹, SR⁹, NR⁹NHR¹⁰,NR⁹R¹⁰, ═O and ═NR⁹, in which R⁹ and R¹⁰ are members independentlyselected from H, substituted or unsubstituted alkyl, acyl, substitutedor unsubstituted heteroalkyl and substituted or unsubstituted aryl.

[0010] The symbol R1 represents a substituent that is a member selectedfrom H, acyl, substituted or unsubstituted alkyl, OR¹¹, SR¹¹, NR^(11a),NR^(12a), halogen, and ═O. The symbol R¹¹ represents a member selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheterocycloalkyl, or substituted or unsubstituted heteroaryl. R^(11a)and R^(12a) are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl.

[0011] R³ is a member selected from H, acyl, substituted orunsubstituted alkyl, NR¹²R¹³, NR¹²OR¹³, SR¹², (═O) and OR¹². The symbolsR¹² and R¹³ represent members independently selected from H, substitutedor unsubstituted alkyl, acyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl or substituted or unsubstitutedheteroaryl.

[0012] R⁴ and R^(4a) are members independently selected from H, halogen,OMe and OH. In a preferred embodiment, the halogen is F.

[0013] R⁵ and R⁶ are members independently selected from H, and OR¹⁴.The symbol R¹⁴ represents H, substituted or unsubstituted alkyl, acyl,substituted or unsubstituted heteroalkyl, or substituted orunsubstituted aryl and P(O)(R¹⁵)(R¹⁶). R¹⁵ and R¹⁶ are independentlyselected from OR¹⁷, NR¹⁷R¹⁸, substituted or unsubstituted alkyl andsubstituted or unsubstituted nucleosides. R¹⁷ and R¹⁸ are independentlyselected from H, CH₂CH CN, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl and substituted or unsubstituted heteroaryl.

[0014] A member selected from R⁵ and R³; R⁶ and R³; and R¹⁵ and R¹⁶together with the atoms to which they are attached, are optionallyjoined to form a ring system selected from substituted or unsubstitutedcycloalkyl and substituted or unsubstituted heterocycloalkyl. In anexemplary embodiment, the ring system is a 5 or 6 membered ring system.

[0015] R⁷ and R⁸ are independently selected from H, acyl, substituted orunsubstituted alkyl. R¹ and R⁸, together with the atoms to which theyare attached are optionally joined into a ring system selected fromsubstituted or unsubstituted cycloalkyl and substituted or unsubstitutedheterocycloalkyl.

[0016] In another aspect of the present invention, the nucleoside andnucleotide analogues (e.g., the compounds of Formula I) of the presentinvention are used for treating a viral disease by administering atherapeutically effective amount of a compound of Formula I to a patientwith a viral disease. In some embodiments, the compounds are givenorally. In other embodiments, the compound is given in an entericformulation. In a further embodiment, the compound is delivered in anoral osmotic drug delivery device.

[0017] When the compounds are given orally, it is generally preferredthat they have a bioavailability that is greater than about 15%, morepreferably greater than about 20% of the administered dose. In anexemplary embodiment, the compound is formulated as an acid additionsalt, e.g. a quaternary ammonium salt. The salt is generally formed bycontacting the compound with a mineral or an organic acid. In apreferred embodiment, the acid is a carboxylic acid, such as palmiticacid.

[0018] The viral disease can be a viral disease caused by an RNA virus,a DNA virus, or a retrovirus. In some embodiments, the viral disease iscaused by HIV. In a further embodiment, the HIV strain is resistant tonucleotide reverse transcriptase inhibitors or other treatments of HIVinfection, including non-nucleoside reverse transcriptase inhibitors, orprotease inhibitors. In other embodiments, the viral disease is causedby a virus of the Flaviviridae family. In a further embodiment the viraldisease is hepatitis C. In other embodiments, the viral disease iscaused by a virus of the Paramyxoviridae family. In a further aspect,the virus is hepatitis B virus or smallpox/vaccinia virus.

[0019] In another aspect of the present invention the compounds ofFormula I are used to treat treat cancer, e.g., hematopoetic cancers.

[0020] Other aspects, objects and advantages of the present inventionwill be apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is an illustration of the hydrophobic-hydrophobic stackinginteractions of selected compositions of the invention.

[0022]FIG. 2 is an illustration of the complexes of the invention formedbetween the pharmacophore modified with a hydrophobic modifying groupand a poly-ion.

[0023]FIG. 3 is an illustration of the complexes of the invention formedbetween the pharmacophore modified with a hydrophobic modifying groupand a dendrimeric poly-ion.

[0024]FIG. 4 depicts the EC₅₀ values for 5-Aza-dC, DHAdC and 5-Me-DHAdCagainst wild-type HIV virus. The experiments were carried out in MT-2cells infected with HIV strain LAI.

[0025]FIG. 5 is an illustration of compounds 1-4.

[0026]FIG. 6 is an exemplary synthetic scheme for compounds 7 and 8.

[0027]FIG. 7 is an exemplary synthetic scheme for compounds 9 and 10.

[0028]FIG. 8 is an exemplary synthetic scheme for compounds 9, and11-14.

[0029]FIG. 9 is an exemplary synthetic scheme for compounds 14-18.

[0030]FIG. 10 is an exemplary synthetic scheme for compounds 20-21.

[0031]FIG. 11 is an exemplary synthetic scheme for compounds 20-21.

[0032]FIG. 12 is an exemplary synthetic scheme for a compound of theinvention including a modified phosphodiester group.

[0033]FIG. 13 is an exemplary synthetic scheme for a compound of theinvention including a modified phosphodiester group derivatized with ahydrophobic moiety.

[0034]FIG. 14 is a retrosynthetic scheme for preparing a compound of theinvention.

[0035]FIG. 15 is an exemplary synthetic scheme for compounds 23, 24 and26.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Introduction

[0037] The present invention is directed to a method of inducing viralmutagenesis, using hydrolytically stable derivatives and formulations of5-aza-cytidine, 5-aza-2′-deoxycytidine and derivatives and variantsthereof, which is useful in cell culture as well as in therapy foranimals and humans. This method is advantageous in that it is usefulagainst DNA or RNA viruses (i.e., viruses that have DNA or RNA genomes).In one embodiment, the methods of the invention are advantageous whenused to target RNA viruses (viruses with an RNA genome), andretroviruses or other viruses otherwise replicated by an RNAintermediate. In another embodiment, the methods of the invention areadvantageous for targeting DNA viruses such as hepatitis B virus, herpesviruses, and papilloma viruses. Without being held to a mechanism ofaction, in one embodiment, the methods of the invention utilizemiscoding nucleosides and nucleotides that are incorporated into bothviral encoded and cellular encoded viral genomic nucleic acids, therebycausing miscoding in progeny copies of the genomic virus, e.g., bytautomerism, which promotes base mispairing (see, e.g., Moriyama et al.,Nucleic Acids Symposium 42: 131-132 (1999); Robinson et al.,Biochemistry 37: 10897-10905 (1998); Anensen et al., Mutation Res. 476:99-107 (2001); Lutz et al., Bioorganic & Medicinal Chem. Letts. 8:499-504 (1998); and Klungland et al., Toxicology Letts. 119: 71-78(2001)).

[0038] The virus may be one in which the viral genomic nucleic acid isintegrated into the cellular genome. Examples of viruses that integratetheir cellular genome include, but are not limited to, retroviruses. Inone particularly preferred embodiment, the virus is HIV. Other preferredviruses include HIV-1, HIV-2, HTLV-1, HTLV-II, and SIV. In anotherembodiment, the virus is a DNA virus such as hepatitis B virus, herpesviruses (e.g., HSV, CMV, EBV), smallpox virus, or papilloma virus (e.g.,HPV). Alternatively, the viral genome can be episomal. These includemany human and animal pathogens, e.g., flaviviruses such as denguefever, West Nile virus, and yellow fever, pestiviruses (a genus of theFlaviviridae family) such as BVDV (bovine viral diarrhea virus),hepatitis C viruses (also a genus of the Flaviviridae family),filoviruses such as ebola virus, influenza viruses, parainfluenzaviruses, including respiratory syncytial virus, measles, mumps, thepicornaviruses, including the echoviruses, the coxsackieviruses, thepolioviruses, the togaviruses, including encephalitis, coronoviruses,rubella, bunyaviruses, reoviruses, including rotaviruses, rhabdoviruses,arenaviruses such as lymphocytic choriomeningitis as well as other RNAviruses of man and animals.

[0039] Retroviruses that can be targeted include the human T-cellleukemia (HTLV) viruses such as HTLV-1 and HTLV-2, adult T-cell leukemia(ATL), the human immunodeficiency viruses such as HIV-1 and HIV-2 andsimian immunodeficiency virus (SIV). In some embodiments, the HIV virusis resistant to non-nucleoside reverse transcriptase inhibitors. Incertain embodiments, the virus is hepatitis A or hepatitis B. See, e.g.,Fields Virology (3rd ed. 1996). Further information regarding viraldiseases and their replication can be found in White and Fenner, MedicalVirology 4th ed. Academic Press (1994) and in Principles and Practice ofClinical Virology, ed. Zuckerman, Banatvala and Pattison, John Wiley andSons (1994). In addition, the compounds of the invention can be used totreat cancer.

[0040] Assays for detecting the mutagenic potential of a nucleoside ornucleotide analog are provided (see, e.g., Example 1). In the assays,the nucleoside or nucleotide analog is incorporated into a viral nucleicacid in the presence of a nucleic acid template, the nucleic acidsynthesized by a cellular or viral polymerase, and a determination ismade regarding whether the incorporation causes a mutation in a progenyvirus. Optionally, naturally occurring (i.e., G, A, U, and/or C)nucleotides are also incorporated into the nucleic acid polymer. Themethod optionally comprises comparing the rate of incorporation of thenucleoside or nucleotide analog and any naturally occurringribonucleoside in the assay into the nucleic acid. For additionalexamples of assays, see, e.g., U.S. Pat. No. 6,132,776, 6,130,036,6,063,628, and 5,512,431 and patent applications U.S. Ser. No.10/226,799 and 60/314,728, which are incorporated herein by reference intheir entirety.

[0041] Exemplary compounds for use in the methods of the inventioninclude 5-aza-cytidine, 5-aza-2′-deoxycytidine, and derivatives andvariants thereof.

[0042] Definitions

[0043] Where substituent groups are specified by their conventionalchemical formulae, written from left to right, they equally encompassthe chemically identical substituents which would result from writingthe structure from right to left, e.g., —CH₂O— is intended to alsorecite —OCH₂—; —NHS(O)₂— is also intended to represent. —S(O)₂HN—, etc.

[0044] As used herein, “linking member” refers to an alkylene unit or acovalent chemical bond that includes at least one heteroatom. Exemplarylinking members include —C(O)NH—, —C(O)O—, —NH—, —S—, —O—, and the like.

[0045] The term “targeting group” is intended to mean a moiety that is(1) able to direct the entity to which it is attached (e.g., therapeuticagent or marker) to a target region, e.g. cell; or (2) is preferentiallyactivated at a target region, for example a region of viral infection.The targeting group can be a small molecule, which is intended toinclude both non-peptides and peptides. The targeting group can also bea macromolecule, which includes saccharides, lectins, receptors, ligandfor receptors, proteins such as BSA, antibodies, and so forth.

[0046] The term “alkyl,” by itself or as part of another substituent,means, unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or poly-unsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups, whichare limited to hydrocarbon groups, are termed “homoalkyl”.

[0047] The term “alkylene” by itself or as part of another substituentmeans a divalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

[0048] The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy)are used in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

[0049] “Acyl” refers to a moiety that is a residue of a carboxylic acidfrom which an oxygen atom is removed, i.e., —C(O)R, in which R issubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl.

[0050] The term “heteroalkyl,” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and at least oneheteroatom selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N and S and Si may be placed at any interior position of the heteroalkylgroup or at the position at which the alkyl group is attached to theremainder of the molecule. Examples include, but are not limited to,—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both (O)₂R′— and —R′C(O)₂—.

[0051] The terms “cycloalkyl” and “heterocycloalkyl”, by themselves orin combination with other terms, represent, unless otherwise stated,cyclic versions of “alkyl” and “heteroalkyl”, respectively.Additionally, for heterocycloalkyl, a heteroatom can occupy the positionat which the heterocycle is attached to the remainder of the molecule.Examples of cycloalkyl include, but are not limited to, cyclopentyl,cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.Examples of heterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

[0052] The term “aryl” means, unless otherwise stated, apolyunsaturated, typically aromatic, hydrocarbon substituent, which canbe a single ring or multiple rings (up to three rings), which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from zero to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

[0053] For brevity, the term “aryl” when used in combination with otherterms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl andheteroaryl rings as defined above. Thus, the term “arylalkyl” is meantto include those radicals in which an aryl group is attached to an alkylgroup (e.g., benzyl, phenethyl, pyridylmethyl and the like) includingthose alkyl groups in which a carbon atom (e.g., a methylene group) hasbeen replaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0054] The terms “halo” or “halogen,” by themselves or as part ofanother substituent, mean, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom. Additionally, terms such as“haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. Forexample, the term “halo(C₁-C₄)alkyl” is mean to include, but not belimited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

[0055] Substituents for the alkyl and heteroalkyl radicals (includingthose groups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′-C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″ R′″)═NR″″,—NR—C(NR′R″)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

[0056] Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″,—SR′, -halogen, —SiR′R″ R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system. When a compound of the invention includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″ and R″″ groups when more than one ofthese groups is present.

[0057] Two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—,—O—, —CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)-B-, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

[0058] As used herein, the term “heteroatom” includes oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

[0059] “Moiety” refers to the radical of a molecule that is attached toanother moiety.

[0060] The symbol “R” is a general abbreviation that represents asubstituent group that is selected from substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted heterocyclyl groups.

[0061] “Reactive functional group,” as used herein refers to groupsincluding, but not limited to, olefins, acetylenes, alcohols, phenols,ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters,amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids,isonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids, thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds. Reactive functional groups alsoinclude those used to prepare bioconjugates, e.g., N-hydroxysuccinimideesters, maleimides and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).

[0062] “Protecting group,” as used herein refers to a portion of asubstrate that is substantially stable under a particular reactioncondition, but which is cleaved from the substrate under a differentreaction condition. A protecting group can also be selected such that itparticipates in the direct oxidation of the aromatic ring component ofthe compounds of the invention. For examples of useful protectinggroups, see, for example, Greene et al., PROTECTIVE GROUPS IN ORGANICSYNTHESIS, John Wiley & Sons, New York, 1991.

[0063] As used herein the term “nucleoside,” includes both the naturallyoccurring nucleosides and modifications thereof. Modifications include,but are not limited to, those providing chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, and electrostaticinteraction to the nucleosides. Such modifications include, but are notlimited to, peptide nucleic acids (PNAs), 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, isobases, such as isocytidine andisoguanidine and the like. “Nucleosides” can also include non-naturalbases, such as, for example, nitroindole. Modifications can also includederivitization with a quencher, a fluorophore or another moiety.“Nucleotides” are phosphate esters of nucleosides. Many modifications ofnucleosides can be also be practiced on nucleotides.

[0064] The symbol

, whether utilized as a bond or displayed perpendicular to a bondindicates the point at which the displayed moiety is attached to theremainder of the molecule, solid support, etc.

[0065] The term “pharmaceutically acceptable salts” includes salts ofthe active compounds prepared with relatively nontoxic acids or bases,depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, palmitic and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

[0066] The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

[0067] In addition to salt forms, the present invention providescompounds, which are in a prodrug form. Prodrugs of the compoundsdescribed herein are those compounds that readily undergo chemicalchanges under physiological conditions to provide the compounds of thepresent invention. Additionally, prodrugs can be converted to thecompounds of the present invention by chemical or biochemical methods inan ex vivo environment. For example, prodrugs can be slowly converted tothe compounds of the present invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent.

[0068] Certain compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

[0069] Certain compounds of the present invention possess asymmetriccarbon atoms (optical centers) or double bonds; the racemates,diastereomers, geometric isomers and individual isomers are encompassedwithin the scope of the present invention.

[0070] The compounds of the invention may be prepared as a single isomer(e.g., enantiomer, cis-trans, positional, diastereomer) or as a mixtureof isomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

[0071] The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

[0072] The term “viral disease” refers to a condition caused by a virus.A viral disease can be caused by a DNA virus, an RNA virus, or by aretrovirus. In some embodiments, viral diseases include virus of theFlavaviridae family. The family Flaviviridae includes the three generaof the family, the flaviviruses, the pestiviruses (e.g., BVDV), and thehepatitis C viruses (e.g., HCV). The family Paramyxoviridae includeswithout limitation, parainfluenza virus, respiratory syncytial virus,Newcastle Disease virus, mumps virus and measles virus. DNA virusincludes the family Poxyiridae. Poxyiridae family members includevaccinia virus and variola virus, which can cause small pox. DNA virusincludes, but is not limited to, the Hepatitis B virus, which replicatesits genome through an RNA intermediate. Retrovirus includes HIV-1,HIV-2, HTLV-1, HTLV-II, and SIV.

[0073] In a preferred embodiment, the compounds of the invention areused to treat n HIV strain that is resistant to nucleoside reversetranscriptase inhibitors (NRTI).

[0074] The four “naturally occurring nucleotides” in RNA and DNA containadenine, guanine, uracil, thymine or cytosine. Nucleotides which arecomplementary to one another are those that tend to form complementaryhydrogen bonds between them and, specifically, the natural complement toA is U or T, the natural complement to U is A, the natural complement toT is A, the natural complement to C is G and the natural complement to Gis C.

[0075] A “nucleic acid” is a deoxyribonucleotide or ribonucleotidepolymer in either single- or double-stranded form, and unless otherwiselimited, encompasses analogs of natural nucleotides.

[0076] A “nucleoside analog” as used herein is defined in more detailbelow and includes analogs of ribonucleosides and deoxyribonucleosidesand the mono- di-, an triphosphates (nucleotides) thereof. As describedabove, they can be naturally occurring or non-naturally occurring, andderived from natural sources or synthesized. These monomeric units arenucleoside analogs (or “nucleotide” analogs if the monomer is consideredwith reference to phosphorylation). For instance, structural groups areoptionally added to the sugar or base of a nucleoside for incorporationinto an oligonucleotide, such as a methyl or allyl group at the 2′-Oposition on the sugar, or a fluoro group which substitutes for the 2′-Ogroup, or a bromo group on the nucleoside base. The phosphodiesterlinkage, or “sugar-phosphate backbone” of the oligonucleotide analog issubstituted or modified, for instance with methyl phosphonates orO-methyl phosphates.

[0077] A “genomic nucleic acid” is a nucleic acid polymer homologous toa nucleic acid which encodes a naturally occurring nucleic acid polymer(RNA or DNA) packaged by a viral particle. Typically, the packagednucleic acid encodes some or all of the components necessary for viralreplication. The genomic nucleic acid optionally includes nucleotideanalogs. Nucleic acids are homologous when they are derived from anucleic acid with a common sequence (an “ancestral” nucleic acid) bynatural or artificial modification of the ancestral nucleic acid.Retroviral genomic nucleic acids optionally encode an RNA competent tobe packaged by a retroviral particle. Such nucleic acids can beconstructed by recombinantly combining a packaging site with a nucleicacid of choice.

[0078] A “virally infected cell” is a cell transduced with a viralnucleic acid. The nucleic acid is optionally incorporated into thecellular genome, or is optionally episomal.

[0079] The “mutation rate” of a virus or nucleic acid refers to thenumber of changes occurring upon copying the nucleic acid, e.g., by apolymerase. Typically, this is measured over time, i.e., the number ofalterations occurring during rounds of copying or generations of virus.

[0080] A “polymerase” refers to an enzyme (DNA or RNA polymerase) thatproduces a polynucleotide sequence, complementary to a pre-existingtemplate polynucleotide (DNA or RNA). For example, an RNA polymerase maybe either an RNA viral polymerase or replicase or RNA cellularpolymerase. A “cellular polymerase” is a polymerase derived from a cell.The cell may be prokaryotic or eukaryotic. The cellular RNA polymeraseis typically an RNA polymerase such as Pol II or Pol III. Pol II enzymesare most preferred. A “mammalian RNA polymerase II” is an RNA polymeraseII derived from a mammal. A “human RNA polymerase II” is an RNApolymerase II derived from a human. A “murine RNA polymerase II” is anRNA polymerase II derived from a mouse. The polymerase is optionallynaturally occurring, or artificially (e.g., recombinantly) produced.

[0081] A “cell culture” is a population of cells residing outside of ananimal. These cells are optionally primary cells isolated from a cellbank, animal, or blood bank, or secondary cells cultured from one ofthese sources, or long-lived artificially maintained in vitro culturesthat are widely available.

[0082] A “progressive loss of viability” refers to a measurablereduction in the replicative or infective ability of a population ofviruses over time.

[0083] A “viral particle” is a viral particle substantially encoded byan RNA virus or a virus with an RNA intermediate, such as BVDV, HCV, orHIV. The presence of non-viral or cellular components in the particle isa common result of the replication process of a virus, which typicallyincludes budding from a cellular membrane.

[0084] An “HIV particle” is a retroviral particle substantially encodedby HIV. The presence of non-HIV viral or cellular components in theparticle is a common result of the replication process of HIV, typicallyincluding budding from a cellular membrane. In certain applications,retroviral particles are deliberately “pseudotyped” by co-expressingviral proteins from more than one virus (often HIV and VSV) to expandthe host range of the resulting retroviral particle. The presence orabsence of non-HIV components in an HIV particle does not change theessential nature of the particle, i.e., the particle is still producedas a primary product of HIV replication.

[0085] Where the methods discussed below require sequence alignment,such methods of alignment of sequences for comparison are well known inthe art. Optimal alignment of sequences for comparison may be conductedby the local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2: 482; by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48: 443; by the search for similarity methodof Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; bycomputerized implementations of these algorithms (including, but notlimited to CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis., USA); the CLUSTAL program is well described by Higginsand Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS5: 151-153; Corpet, et al., (1988) Nucleic Acids Research 16, 10881-90;Huang, et al., (1992) Computer Applications in the Biosciences 8,155-65, and Pearson, et al., (1994) Methods in Molecular Biology 24,307-31. Typically, the alignments are visually inspected and refinedmanually after computer-aided adjustment.

[0086] A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the defaultparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

[0087] As used herein, “cancer” includes solid tumors and hematologicalmalignancies. The former includes cancers such as breast, colon, andovarian cancers. The latter include hematopoietic malignancies includingleukemias, lymphomas and myelomas. This invention provides new effectivemethods, compositions, and kits for treatment and/or prevention ofvarious types of cancer.

[0088] Hematological malignancies, such as leukemias and lymphomas, areconditions characterized by abnormal growth and maturation ofhematopoietic cells.

[0089] Leukemias are generally neoplastic disorders of hematopoieticstem cells, and include adult and pediatric acute myeloid leukemias(AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL), hairy cell leukemia and secondaryleukemia. Myeloid leukemias are characterized by infiltration of theblood, bone marrow, and other tissues by neoplastic cells of thehematopoietic system. CLL is characterized by the accumulation ofmature-appearing lymphocytes in the peripheral blood and is associatedwith infiltration of bone marrow, the spleen and lymph nodes.

[0090] Specific leukemias include acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemicleukemia, a leukocythemic leukemia, basophylic leukemia, blast cellleukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis,embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cellleukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocyticleukemia, stem cell leukemia, acute monocytic leukemia, leukopenicleukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocyticleukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cellleukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblasticleukemia, monocytic leukemia, myeloblastic leukemia, myelocyticleukemia, myeloid granulocytic leukemia, myelomonocytic leukemia,Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia,promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stemcell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0091] Lymphomas are generally neoplastic transformations of cells thatreside primarily in lymphoid tissue. Among lymphomas, there are twomajor distinct groups: non-Hodgkin's lymphoma (NHL) and Hodgkin'sdisease. Lymphomas are tumors of the immune system and generally arepresent as both T cell- and as B cell-associated disease. Bone marrow,lymph nodes, spleen and circulating cells are all typically involved.Treatment protocols include removal of bone marrow from the patient andpurging it of tumor cells, often using antibodies directed againstantigens present on the tumor cell type, followed by storage. Thepatient is then given a toxic dose of radiation or chemotherapy and thepurged bone marrow is then reinfused in order to repopulate thepatient's hematopoietic system.

[0092] Other hematological malignancies include myelodysplasticsyndromes (MDS), myeloproliferative syndromes (MPS) and myelomas, suchas solitary myeloma and multiple myeloma. Multiple myeloma (also calledplasma cell myeloma) involves the skeletal system and is characterizedby multiple tumorous masses of neoplastic plasma cells scatteredthroughout that system. It may also spread to lymph nodes and othersites such as the skin. Solitary myeloma involves solitary lesions thattend to occur in the same locations as multiple myeloma.

[0093] Hematological malignancies are generally serious disorders,resulting in a variety of symptoms, including bone marrow failure andorgan failure. Treatment for many hematological malignancies, includingleukemias and lymphomas, remains difficult, and existing therapies arenot universally effective. While treatments involving specificimmunotherapy appear to have considerable potential, such treatments arelimited by the small number of known malignancy-associated antigens.Moreover the ability to detect such hematological malignancies in theirearly stages can be quite difficult depending upon the particularmalady. Accordingly, there remains a need in the art for improvedmethods for treatment of hematological malignancies such as B cellleukemias and lymphomas and multiple myelomas. The present inventionfulfills these and other needs in the field.

[0094] Other cancers are also of concern, and represent similardifficulties insofar as effective treatment is concerned. Such cancersinclude those characterized by solid tumors. Examples of other cancersof concern are skin cancers, including melanomas, basal cell carcinomas,and squamous cell carcinomas. Epithelial carcinomas of the head and neckare also encompassed by the present invention. These cancers typicallyarise from mucosal surfaces of the head and neck and include salivarygland tumors.

[0095] The present invention also encompasses cancers of the lung. Lungcancers include squamous or epidermoid carcinoma, small cell carcinoma,adenocarcinoma, and large cell carcinoma. Breast cancer is alsoincluded, both invasive breast cancer and non-invasive breast cancer,e.g., ductal carcinoma in situ and lobular neoplasia.

[0096] The present invention also encompasses gastrointestinal tractcancers. Gastrointestinal tract cancers include esophageal cancers,gastric adenocarcinoma, primary gastric lymphoma, colorectal cancer,small bowel tumors and cancers of the anus. Pancreatic cancer andcancers that affect the liver are also of concern, includinghepatocellular cancer. The present invention also includes treatment ofbladder cancer and renal cell carcinoma.

[0097] The present invention also encompasses prostatic carcinoma andtesticular cancer.

[0098] Gynecologic malignancies are also encompassed by the presentinvention including ovarian cancer, carcinoma of the fallopian tube,uterine cancer, and cervical cancer.

[0099] Treatment of sarcomas of the bone and soft tissue are encompassedby the resent invention. Bone sarcomas include osteosarcoma,chondrosarcoma, and Ewing's sarcoma.

[0100] The present invention also encompasses malignant tumors of thethyroid, including papillary, follicular, and anaplastic carcinomas.

[0101] In some embodiments, a “subject in need of treatment” is a mammalwith a viral disease that is life-threatening, or that impairs health,or shortens the lifespan of the mammal. In other embodiments, a “subjectin need of treatment” is a mammal with cancer that is life-threateningor that impairs health or shortens the lifespan of the mammal.

[0102] A “pharmaceutically acceptable” component is one that is suitablefor use with humans and/or animals without undue adverse side effects(such as toxicity, irritation, and allergic response) commensurate witha reasonable benefit/risk ratio.

[0103] A “safe and effective amount” refers to the quantity of acomponent that is sufficient to yield a desired therapeutic responsewithout undue adverse side effects (such as toxicity, irritation, orallergic response) commensurate with a reasonable benefit/risk ratiowhen used in the manner of this invention. In some embodiments“therapeutically effective amount” refers to an amount of a componenteffective to yield the desired therapeutic response, for example, anamount effective to enhance mutagenesis of a virus, or to diminish theability of the virus to produce active proteins, or to inhibitreplication of a virus, or to eliminate or diminish the ability of avirus to produce infectious particles, or to kill the virus or a virallyinfected cell. Other embodiments encompass other therapeutic responses,for example, an amount of a component effective to halt or to delay thegrowth of a cancer, or to cause a cancer to shrink, or not metastasize.The specific safe and effective amount or therapeutically effectiveamount will vary with such factors as the particular condition beingtreated, the physical condition of the patient, the type of mammal beingtreated, the duration of the treatment, the nature of concurrent therapy(if any), and the specific formulations employed and the structure ofthe compounds or its derivatives.

[0104] An “enteric formulation” is a formulation of a compound whereinthe compound is stable in the acidic environment of the stomach, andafter passage through the stomach, an active form of the compound isavailable for absorbtion in the intestinal tract. An “oral osmotic drugdevice” as used herein is a device that delivers a drug at a controlledrate in a region of the gastrointestinal tract having a pH less than3.5, and then delivers all the drug in the immediately continuing regionof the gastrointestinal tract having a pH greater than 3.5. Methods tomake and use an oral osmotic drug device are found, for example, in U.S.Pat. No. 4,587,117, herein incorporated by reference.

[0105] The Compounds

[0106] The present invention provides compounds that display antiviralactivity, in addition to salts and prodrugs of such compounds. Thecompounds are generally nucleosides, nucleotides, nucleoside analogues,nucleotide analogues, salts and prodrugs thereof. The inventors haverecognized that antiviral pharamacophores comprising highly active, yetbiologically unstable nucleosides or nucleotides and nucleoside ornucleotide analogues are converted to useful therapeutic agents byaltering selected properties of the pharmacophore. In an exemplaryembodiment, the pharmacophore is stabilized by the attachment of theactive species containing the pharmacophore to a modifying group thatincreases the lipophilicity of the pharmacophore. The combination of thepharmacophore and the modifying group preferably provides thepharmacophore in a prodrug format.

[0107] Prodrugs comprise inactive forms of active drugs in which achemical group is present on the prodrug, which renders it inactiveand/or confers solubility or some other property to the drug. Prodrugsare generally inactive, or less active than the parent compound, butonce the chemical group has been cleaved from the prodrug (e.g., byhydrolysis, heat, cavitation, pressure, and/or enzymes in thesurrounding environment), the active drug is generated. Prodrugs may bedesigned as reversible drug derivatives and utilized as modifiers toenhance drug transport to site-specific tissues. Prodrugs are describedin the art, for example, in Sinkula et al., J. Pharm. Sci 64: 181-210(1975) and in U.S. Provisional Patent Application No. 60/480,037, filedJun. 20, 2003, which is herein incorporated by reference for allpurposes.

[0108] Thus, the present invention provides, inter alia, novelnucleoside and nucleotide analogues that are covalently attached to agroup that modifies the properties of the nucleoside or nucleotideanalogue. In exemplary embodiments, the “modifying group” enhances thestability or bioavailability of nucleoside or nucleotide or itsanalogue. In the discussion that follows, the invention is exemplifiedby reference to lipophilic modifying groups. The focus of the discussionis for clarity of illustration, and those of skill in the art willappreciate that compounds including modifying groups other thelipophilic groups discussed herein are within the scope of theinvention.

[0109] Thus, in a first aspect, there is provided a compound accordingto Formula I:

[0110] In Formula I, the dashed circle indicates that the ring systemmay include one or more double bonds at any position, such that thevalence of the intra-annular atoms is satisfied. The ring system may bearomatic (e.g., heteroaryl) or non-aromatic. The substituents R², R⁷, R⁹are present or absent as dictated by the application of the laws ofvalency to a selected ring structure.

[0111] The symbol Y represents C, CH or N, and the symbol Z representsC, CH or B. R¹ is a member selected from H, acyl, OR⁹, SR⁹, NR⁹NHR¹⁰,NR⁹R¹⁰, ═O and ═NR⁹, in which R⁹ and R10 are members independentlyselected from H, substituted or unsubstituted alkyl, acyl, substitutedor unsubstituted heteroalkyl and substituted or unsubstituted aryl.

[0112] The symbol R² represents a substituent that is a member selectedfrom H, acyl, substituted or unsubstituted alkyl, OR¹¹, SR¹¹, NR^(11a),NR^(12a), halogen, and ═O. The symbol R¹¹ represents a member selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheterocycloalkyl, or substituted or unsubstituted heteroaryl. R^(11a)and R^(12a) are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl.

[0113] R³ is a member selected from H, acyl, substituted orunsubstituted alkyl, NR¹²R¹³, NR¹²OR¹³, SR¹², (═O) and OR¹². The symbolsR¹² and R¹³ represent members independently selected from H, substitutedor unsubstituted alkyl, acyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl or substituted or unsubstitutedheteroaryl.

[0114] R⁴ and R^(4a) are members independently selected from H, halogen,OMe and OH. In a preferred embodiment, the halogen is F.

[0115] R⁵ and R⁶ are members independently selected from H, and OR¹⁴.The symbol R¹⁴ represents H, substituted or unsubstituted alkyl, acyl,substituted or unsubstituted heteroalkyl, or substituted orunsubstituted aryl and P(O)(R¹⁵)(R¹⁶). R¹⁵ and R¹⁶ are independentlyselected from OR¹⁷, NR¹⁷R¹⁸, substituted or unsubstituted alkyl andsubstituted or unsubstituted nucleosides. R¹⁷ and R¹⁸ are independentlyselected from H, CH₂CH CN, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl and substituted or unsubstituted heteroaryl.

[0116] A member selected from R⁵ and R³; R⁶ and R³; and R¹⁵ and R¹⁶together with the atoms to which they are attached, are optionallyjoined to form a ring system selected from substituted or unsubstitutedcycloalkyl and substituted or unsubstituted heterocycloalkyl. In anexemplary embodiment, the ring system is a 5 or 6 membered ring system.

[0117] R⁷ and R⁸ are independently selected from H, acyl, substituted orunsubstituted alkyl. R¹ and R⁸, together with the atoms to which theyare attached are optionally joined into a ring system selected fromsubstituted or unsubstituted cycloalkyl and substituted or unsubstitutedheterocycloalkyl.

[0118] In an exemplary embodiment, the invention provides a compoundaccording to Formula II:

[0119] in which the identity of each of the radicals is substantially asdescribed above.

[0120] In another exemplary embodiment, there is provided a compoundaccording to Formula III:

[0121] In an exemplary compound according to Formula III, R¹¹ iscleaveable moiety, for example, a silyl group or substituted orunsubstituted alkyl ether, e.g.,

[0122] In a still further exemplary embodiment, the invention provides acompound of Formula IV:

[0123] Exemplary compounds according to the Formulae above include:

[0124] Still further exemplary compounds based upon apolynucleotide-like format include:

[0125] In a further embodiment, the present invention provides acompound according to Formula V:

[0126] in which R¹⁹, R²⁰, and R²¹ are members independently selectedfrom H, acyl and substituted or unsubstituted alkyl.

[0127] Compounds according to Formula V, provide the active compound byelimination of the nitrogen “protecting group”:

[0128] R═H, OH; R′ is a leaving group OAlkyl, OAryl, OHeteroaryl,SAlkyl, SAryl, S(O)Heteroaryl, S(O)₂Heteroaryl, S(O)₂Alkyl, S(O)Aryl,S(O)₂Heteroaryl, Cl, Br, 1, N(Alkyl)₂; R″, R′″, and R″″ are nitrogenprotecting groups

[0129] In an exemplary embodiment, R⁶ has a structure according toFormula VI or Formula VII:

[0130] in which R²² represents substituted or unsubstituted alkyl or asubstituted or unsubstituted heteroalkyl moiety. The symbol L representsa linker selected from substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl; and Ar is a member selectedfrom substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. The symbol n represents an integer from 1 to 30.

[0131] An exemplary linker precursor contains at least two linkinggroups derived from reactive functional groups. Typically, one linkinggroup of the linker bonds to an oxygen of the phosphate(phosphodiester), while the other linking group of the linker bonds to achemical functionality of the pharmaceutical agent. Examples of chemicalfunctionalities of linker groups include hydroxy, mercapto, carbonyl,carboxy, amino, ketone, and mercapto groups.

[0132] Exemplary linker groups include 6-aminohexanol,6-mercaptohexanol, 10-hydroxydecanoic acid, glycine and other aminoacids, 1,6-hexanediol, α-alanine, 2-aminoethanol, cysteamine(2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid,3-maleimidobenzoic acid, phthalide, α-substituted phthalides, thecarbonyl group, aminal esters, and the like. Other “bifunctional” linkergroups include, but are not limited to, moieties such as sugars (e.g.,polyol with reactive hydroxyl), amino acids, amino alcohols, carboxyalcohols, amino thiols, and the like.

[0133] Generally, at least one of the chemical functionalities of thelinker group, the modifying group or the pharmacophore will be activatedto allow for the formation of the pharmacophore-linker-modifying groupcomplex. One skilled in the art will appreciate that a variety ofchemical functionalities, including hydroxy, amino, and carboxy groups,can be activated using a variety of standard methods and conditions. Forexample, a hydroxyl group of the linker or pharmacophore can beactivated through treatment with phosgene to form the correspondingchloroformate, or p-nitrophenylchloroformate to form the correspondingcarbonate.

[0134] In an exemplary embodiment, the compound of the inventionincludes a linker that includes a carboxyl functionality. Carboxylgroups may be activated by, for example, conversion to the correspondingacyl halide, imidazolide or active ester. This reaction may be performedunder a variety of conditions as illustrated in March, supra pp. 388-89.In a preferred embodiment, the acyl halide is prepared through thereaction of the carboxyl-containing group with oxalyl chloride. Those ofskill in the art will appreciate that the use of carboxyl-containingagents is merely illustrative, and that agents having many otherfunctional groups can be incorporated within the compounds of theinvention.

[0135] Typically, the compounds of the invention are prepared usingstandard chemical techniques to join the various components throughtheir respective chemical functionalities. Those of skill in the artwill recognize that one can first attach the linker either to thepharmacophore or to the modifying group. The exemplary chemicalfunctionalities shown in Table 1 can be present on the pharmacophore,linker, or modifying group, depending on the synthesis scheme employed.Table 1 provides examples of a first chemical functionality that is acomponent of either the pharmacaphore or a substuituent and a secondchemical functionality that is a component of either the pharmacaphoreor a substuituent. The exemplary linkages set forth in Table 1 areproduced by the covalent interaction of chemical functionality 1 and 2.

[0136] The groups set forth in Table 1 are also generally representativeof “active groups,” which are found on core moieties of use in thepresent invention. TABLE 1 Chemical Chemical Functionality 1Functionality 2 Linkage Hydroxy Carboxy Ester Hydroxy Carbonate AmineCarbamate SO₃ Sulfate PO₃ Phosphate Carboxy Acyloxyalkyl Ketone KetalAldehyde Acetal Hydroxy Anhydride Mercapto Mercapto Disulfide CarboxyAcyloxyalkyl Thioether Carboxy Thioester Carboxy Amino amide MercaptoThioester Carboxy Acyloxyalkyl ester Carboxy Acyloxyalkyl amide AminoAcyloxyalkoxy carbonyl Carboxy Anhydride Carboxy N-acylamide HydroxyEster Hydroxy Hydroxymethyl ketone ester Hydroxy Alkoxycarbonyl oxyalkylAmino Carboxy Acyloxyalkylamine Carboxy Acyloxyalkylamide Amino UreaCarboxy Amide Carboxy Acyloxyalkoxycarbonyl Amide N-Mannich base CarboxyAcyloxyalkyl carbamate Phosphate Hydroxy Phosphate oxygen ester AminePhosphoramidate Mercapto Thiophosphate ester Ketone Carboxy Enol esterSulfonamide Carboxy Acyloxyalkyl sulfonamide Ester N-sulfonyl- imidate

[0137] One skilled in the art will readily appreciate that many of theselinkages may be produced in a variety of ways and using a variety ofconditions. For the preparation of esters, see, e.g., March supra at1157; for thioesters, see, March, supra at 362-363, 491, 720-722, 829,941, and 1172; for carbonates, see, March, supra at 346-347; forcarbamates, see, March, supra at 1156-57; for amides, see, March supraat 1152; for ureas and thioureas, see, March supra at 1174; for acetalsand ketals, see, Greene et al. supra 178-210 and March supra at 1146;for acyloxyalkyl derivatives, see, PRODRUGS: TOPICAL AND OCULAR DRUGDELIVERY, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; forenol esters, see, March supra at 1160; for N-sulfonylimidates, see,Bundgaard et al., J. Med. Chem., 31:2066 (1988); for anhydrides, see,March supra at 355-56, 636-37, 990-91, and 1154; for N-acylamides, see,March supra at 379; for N-Mannich bases, see, March supra at 800-02, and828; for hydroxymethyl ketone esters, see, Petracek et al. Annals NYAcad. Sci., 507:353-54 (1987); for disulfides, see, March supra at 1160;and for phosphonate esters and phosphonanidates, see, e.g., copendingapplication Ser. No. 07/943,805, which is expressly incorporated hereinby reference.

[0138] In certain embodiments, one or more of the active groups areprotected during one or more steps of the reaction to assemble thecompound of the invention. Those of skill in the art understand how toprotect a particular functional group such that it does not interferewith a chosen set of reaction conditions. For examples of usefulprotecting groups, see, for example, Greene et al., PROTECTIVE GROUPS INORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

[0139] The linker can also serve to introduce additional molecular massand chemical functionality into the compound of the invention.Generally, the additional mass and functionality will affect the serumhalf-life and other properties of the compound. Thus, through carefulselection of linker groups, compounds of the invention with a range ofserum half-lives can be produced.

[0140] In another exemplary embodiment, the linker includes a bond thatrenders the compound of the invention susceptible to in vivodegradation. In a preferred embodiment, the bond is reversible (e.g.,easily hydrolyzed) or partially reversible (e.g., partially or slowlyhydrolyzed). Cleavage of the bond can occur through biological orphysiological processes. In other embodiments, the physiologicalprocesses will cleave bonds at other locations within the complex (e.g.,removing an ester group or other protecting group that is coupled to anotherwise sensitive chemical functionality) before cleaving the bondbetween the agent and dendrimer, resulting in partially degradedcomplexes. Other cleavages can also occur, for example, between thespacer and agent and the spacer and dendrimer.

[0141] For rapid degradation of the complex after administration,circulating enzymes in the plasma can be used to cleave the dendrimerfrom the pharmaceutical agent. These enzymes can include non-specificaminopeptidases and esterases, dipeptidyl carboxy peptidases, proteasesof the blood clotting cascade, and the like.

[0142] Alternatively, cleavage may occur through nonenzymatic processes.For example, chemical hydrolysis may be initiated by differences in pHexperienced by the complex following delivery. In such a case, thepharmaceutical agent-dendrimer complex may be characterized by a highdegree of chemical lability at physiological pH of 7.4, while exhibitinghigher stability at an acidic or basic pH in the reservoir of thedelivery device. An exemplary pharmaceutical agent-dendrimer complex,which is cleaved in such a process is a complex incorporating aN-Mannich base linkage within its framework.

[0143] In most cases, cleavage of the compound will occur during orshortly after administration. However, in certain embodiments, cleavagedoes not occur until the complex reaches the pharmaceutical agent's siteof action.

[0144] The susceptibility of the compound of the invention todegradation can be ascertained through studies of the hydrolytic orenzymatic conversion of the complex to the unbound pharmaceutical agent.Generally, good correlation between in vitro and in vivo activity isfound using this method. See, e.g., Phipps et al., J. Pharm. Sciences78:365 (1989). The rates of conversion may be readily determined, forexample by spectrophotometric methods or by gas-liquid or high-pressureliquid chromatography. Half-lives and other kinetic parameters may thenbe calculated using standard techniques. See, e.g., Lowry et al.MECHANISM AND THEORY IN ORGANIC CHEMISTRY, 2nd Ed., Harper & Row,Publishers, New York (1981).

[0145] In a preferred embodiment, one or more of the substituents(modifying groups) on the nucleoside or nucleotide (or analogue) core isa lipid, or is lipophilic; an embodiment of the invention that isillustrated by reference to compounds of the invention in which thesubstituent is a hydrophobic species, such as a lipid.

[0146] A wide variety of lipids may be used in preparing thecompositions of the invention. The lipids may be of either natural,synthetic or semi-synthetic origin, including for example, fatty acids,fatty alcohols, neutral fats, phosphatides, oils, glycolipids,surface-active agents (surfactants), aliphatic alcohols, waxes, terpenesand steroids.

[0147] Exemplary lipids which may be used to prepare the compounds ofthe present invention include, for example, fatty acids, lysolipids,fluorolipids, phosphocholines, such as those associated with plateletactivation factors (PAF) (Avanti Polar Lipids, Alabaster, Ala.),including 1-alkyl-2-acetoyl-sn-glycero 3-phosphocholines, andI-alkyl-2-hydroxy-sn-glycero 3-phosphocholines, phosphatidylcholine withboth saturated and unsaturated lipids, includingdioleoylphosphatidylcholine; dimyristoylphosphatidylcholine;dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine;dipalmitoylphosphatidylcholine (DPPC); distearoylphosphatidylcholine(DSPC); and diarachidonylphosphatidylcholine (DAPC);phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine (DPPE) anddistearoylphosphatidylethanolamine (DSPE); phosphatidylserine;phosphatidylglycerols, including distearoylphosphatidyl-glycerol (DSPG);phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipidssuch as ganglioside GM1 and GM2; glucolipids; sulfatides;glycosphingolipids; phosphatidic acids, such as dipalmitoylphosphatidicacid (DPPA) and distearoyl-phosphatidic acid (DSPA); palmitic acid;stearic acid; arachidonic acid; oleic acid; lipids bearing polymers,such as chitin, hyaluronic acid, polyvinyl-pyrrolidone or polyethyleneglycol (PEG), also referred to herein as “pegylated lipids” withpreferred lipid bearing polymers including DPPE-PEG (DPPE-PEG), whichrefers to the lipid DPPE having a PEG polymer attached thereto,including, for example, DPPE-PEG5000, which refers to DPPE havingattached thereto a PEG polymer having a mean average molecular weight ofabout 5000; lipids bearing sulfonated mono-, di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate and cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether andester-linked fatty acids; polymerized lipids (a wide variety of whichare well known in the art); dicetyl phosphate; stearylamine;cardiolipin; phospholipids with short chain fatty acids of about 6 toabout 8 carbons in length; synthetic phospholipids with asymmetric acylchains, such as, for example, one acyl chain of about 6 carbons andanother acyl chain of about 12 carbons; ceramides; non-ionic liposomesincluding niosomes such as polyoxyalkylene (e.g., polyoxyethylene) fattyacid esters, polyoxyalkylene (e.g., polyoxyethylene) fatty alcohols,polyoxyalkylene (e.g., polyoxyethylene) fatty alcohol ethers,polyoxyalkylene sorbitan fatty acid esters (such as, for example, theclass of compounds referred to as TWEEN™, including TWEEN 20, TWEEN 40and TWEEN 80, commercially available from ICI Americas, Inc.,Wilmington, Del.), including polyoxyethylated sorbitan fatty acidesters, glycerol polyethylene glycol oxystearate, glycerol polyethyleneglycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil,polyoxyethylene-polyoxypropylene polymers, and polyoxyethylene fattyacid stearates; sterol aliphatic acid esters including cholesterolsulfate, cholesterol butyrate, cholesterol isobutyrate, cholesterolpalmitate, cholesterol stearate, lanosterol acetate, ergosterolpalmitate, and phytosterol n-butyrate; sterol esters of sugar acidsincluding cholesterol glucuronide, lanosterol glucuronide,7-dehydrocholesterol glucuronide, ergosterol glucuronide, cholesterolgluconate, lanosterol gluconate, and ergosterol gluconate; esters ofsugar acids and alcohols including lauryl glucuronide, stearoylglucuronide, myristoyl glucuronide, lauryl gluconate, myristoylgluconate, and stearoyl gluconate; esters of sugars and aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid and polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, anddigitoxigenin; glycerol dilaurate, glycerol trilaurate, glyceroldipalmitate, glycerol and glycerol esters including glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate; long chain alcohols includingn-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, andn-octadecyl alcohol;6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)-hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside;12-(((7′-diethylamino-coumarin-3-yl)-carbonyl)-methylamino)-octadecanoicacid;N-[12-(((7′-diethylamino-coumarin-3-yl)-carbonyl)-methylamino)-octadecanoy1]-2-aminopalmitic acid; cholesteryl(4′-trimethyl-ammonio)-butanoate;N-succinyldioleoylphosphatidylethanol-amine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycero-phosphoethanolamine andpalmitoylhomocysteine, and/or any combinations thereof.

[0148] Examples of polymerized lipids include unsaturated lipophilicchains such as alkenyl or alkynyl, containing up to about 50 carbonatoms. Further examples are phospholipids such as phosphoglycerides andsphingolipids carrying polymerizable groups, and saturated andunsaturated fatty acid derivatives with hydroxyl groups, such as forexample triglycerides of d-12-hydroxyoleic acid, including castor oiland ergot oil. Polymerization may be designed to include hydrophilicsubstituents such as carboxyl or hydroxyl groups, to enhancedispersability so that the backbone residue resulting frombiodegradation is water-soluble. Suitable polymerizable lipids are alsodescribed, for example, in Klaveness et al, U.S. Pat. No. 5,536,490.

[0149] If desired, the compound of the invention may comprise a cationiclipid, such as, for example,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and1,2-dioleoyl-3-(4′-trimethylammonio)-butanoyl-sn-glycerol (DOTB).

[0150] Exemplary anionic lipids include phosphatidic acid andphosphatidyl glycerol and fatty acid esters thereof, amides ofphosphatidyl ethanolamine such as anandamides and methanandamides,phosphatidyl serine, phosphatidyl inositol and fatty acid estersthereof, cardiolipin, phosphatidyl ethylene glycol, acidic lysolipids,sulfolipids, and sulfatides, free fatty acids, both saturated andunsaturated, and negatively charged derivatives thereof. Phosphatidicacid and phosphatidyl glycerol and fatty acid esters thereof arepreferred anionic lipids.

[0151] Examples of cationic lipids include those listed hereinabove. Apreferred cationic lipid for formation of aggregates isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride(“DOTMA”). Synthetic cationic lipids may also be used. These includecommon natural lipids derivatized to contain one or more basicfunctional groups. Examples of lipids which can be so modified includedimethyldioctadecyl-ammonium bromide, sphinolipids, sphingomyelin,lysolipids, glycolipids such as ganglioside GMI, sulfatides,glycosphingolipids, cholesterol and cholesterol esters and salts,N-succinyldioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol,1,3-dipalmitoyl-2-succinylglycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine andpalmitoylhomocystiene.

[0152] Specially synthesized cationic lipids also function in theembodiments of the invention. Among these are, for example, N,N′-bis(dodecyaminocarbonyl-methylene)-N,N′-bis (β-N,N,N-trimethylammoniumethylaminocarbonylmethylene-ethylene-diamine tetraiodide;N,N″-bis hexadecylaminocarbonylmethylene)-N,N′,N″-tris hexaiodide;N,N′-Bis(dodecylaminocarbonylmethylene)-N,N″-bis(β-N,N,N-trimethylammoniumethylaminocarbonylmethylene)cyclohexylene-1,4-diaminetetraiodide;1,1,7,7-tetra-(β-N,N,N,N-tetramethylammoniumethylaminocarbonylmethylene)-3-hexadecylaminocarbonylmethylene-1,3,7-triaazaheptaneheptaiodide; andN,N,N′N′-tetraphospho-ethanolaminocarbonylmethylene)diethylenetriaminetetraiodide.

[0153] In those embodiments in which both cationic and non-cationiclipids are utilized, a wide variety of lipids, as described above, maybe employed as the non-cationic lipid. Preferably, the non-cationiclipid comprises one or more of DPPC, DPPE anddioleoylphosphatidylethanolamine. In lieu of the cationic lipids listedabove, lipids bearing cationic polymers, such as polylysine orpolyarginine, as well as alkyl phosphonates, alkyl phosphinates, andalkyl phosphites, may also be used in the stabilizing materials. Thoseof skill in the art will recognize, in view of the present disclosure,that other natural and synthetic variants carrying positive chargedmoieties will also function in the invention.

[0154] Saturated and unsaturated fatty acids, which may be employed inthe present compounds, include moieites that preferably contain fromabout 12 carbon atoms to about 22 carbon atoms, in linear or branchedform. Hydrocarbon groups consisting of isoprenoid units and/or prenylgroups can be also used. Examples of suitable saturated fatty acidsinclude, for example, lauric, myristic, palmitic, and stearic acids.Examples of suitable unsaturated fatty acids include, for example,lauroleic, physeteric, myristoleic, palmitoleic, petroselinic, and oleicacids. Examples of suitable branched fatty acids include, for example,isolauric, somyristic, isopalmitic, and isostearic acids.

[0155] Other useful lipids or combinations thereof apparent to thoseskilled in the art, which are in keeping with the spirit of the presentinvention are also encompassed by the present invention. For example,carbohydrate-bearing lipids may be employed, as described in U.S. Pat.No. 4,310,505, the disclosure of which is hereby incorporated herein byreference in its entirety.

[0156] In addition to the lipids set forth above, the compounds of thepresent invention may include a moiety that is derived in whole or inpart, from proteins or derivatives thereof. Suitable proteins for use inthe present invention include, for example, albumin, hemoglobin,α-1-antitrypsin, α-fetoprotein, aminotransferases, amylase, C-reactiveprotein, carcinoembryonic antigen, ceruloplasmin, complement, creatinephosphokinase, ferritin, fibrinogen, fibrin, transpeptidase, gastrin,serum globulins, myoglobin, immunoglobulins, lactate dehydrogenase,lipase, lipoproteins, acid phosphatase, alkaline phosphatase, α-1-serumprotein fraction, α-2-serum protein fraction, β-protein fraction,γ-protein fraction and γ-glutamyl transferase. Other stabilizingmaterials and vesicles formulated from proteins that may be used in thepresent invention are described, for example, in U.S. Pat. Nos.4,572,203, 4,718,433, 4,774,958, and 4,957,656. Other protein-basedmoieties, in addition to those described above and in the aforementionedpatents, are apparent to one of ordinary skill in the art, in view ofthe present disclosure.

[0157] In addition to the lipids and proteins discussed herein,embodiments of the present invention may also include polymers, whichmay be of natural, semi-synthetic (modified natural) or syntheticorigin. Polymer denotes a compound comprised of two or more repeatingmonomeric units, and preferably 10 or more repeating monomeric units.Semi-synthetic polymer (or modified natural polymer) denotes a naturalpolymer that has been chemically modified in some fashion. Examples ofsuitable natural polymers include naturally occurring polysaccharides,such as, for example, arabinans, fructans, fucans, galactans,galacturonans, glucans, mannans, xylans (such as, for example, inulin),levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins,including amylose, pullulan, glycogen, amylopectin, cellulose, dextran,dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin,agarose, keratin, chondroitin, dennatan, hyaluronic acid, alginic acid,xanthin gum, starch and various other natural homopolymer orheteropolymers, such as those containing one or more of the followingaldoses, ketoses, acids or amines: erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose,gulose, idose, galactose, talose, erythrulose, ribulose, xylulose,psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose,sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaricacid, galacturonic acid, mannuronic acid, glucosamine, galactosamine,and neuraminic acid, and naturally occurring derivatives thereof.Accordingly, suitable polymers include, for example, proteins, such asalbumin. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethyl-cellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers suitable for use in the present inventioninclude polyphosphazenes, polyethylenes (such as, for example,polyethylene glycol (including, for example, the class of compoundsreferred to as PLURONICS™, commercially available from BASF, Parsippany,N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes(such as, for example, polypropylene glycol), polyurethanes (such as,for example, polyvinyl alcohol (PVA), polyvinyl chloride andpolyvinylpyrrolidone), polyamides including nylon, polystyrene,polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbonpolymers (such as, for example, polytetrafluoroethylene), acrylate,methacrylate, and polymethylmethacrylate, and derivatives thereof.Preferred are biocompatible synthetic polymers or copolymers preparedfrom monomers, such as acrylic acid, methacrylic acid, ethyleneimine,crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate,2-hydroxyethyl methacrylate (HEMA), lactic acid, glycolic acid,α-caprolactone, acrolein, cyanoacrylate, bisphenol A, epichlorhydrin,hydroxyalkyl-acrylates, siloxane, dimethylsiloxane, ethylene oxide,ethylene glycol, hydroxyalkyl-methacrylates, N-substituted acrylamides,N-substituted methacrylamides, N-vinyl-2-pyrrolidone,2,4-pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene,p-amino-styrene, p-amino-benzyl-styrene, sodium styrene sulfonate,sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethylmethacrylates, 2-methacryloyloxytrimethylammonium chloride, andpolyvinylidene, as well as polyfunctional crosslinking monomers such asN,N′-methylenebisacrylamide, ethylene glycol dimethacrylates,2,2′-(p-phenylenedioxy)-diethyl dimethacrylate, divinylbenzene,triallylamine and methylenebis-(4-phenylisocyanate), includingcombinations thereof. Preferable polymers include polyacrylic acid,polyethyleneimine, polymethacrylic acid, polymethylmethacrylate,polysiloxane, polydimethylsiloxane, polylactic acid,poly(e-caprolactone), epoxy resin, poly(ethylene oxide), poly(ethyleneglycol), and polyamide (nylon) polymers. Preferrred copolymers include,but are not limited to, polyvinylidene-polyacrylonitrile,polyvinylidenepolyacrylonitrile-polymethylmethacrylate,polystyrene-polyacrylonitrile and poly d-1, lactide co-glycolidepolymers. A preferred copolymer is polyvinylidene-polyacrylonitrile.Other suitable biocompatible monomers and polymers will be apparent tothose skilled in the art, in view of the present disclosure.

[0158] In a still further exemplary embodiment, the invention providestricyclic compounds according to Formula VII, in which the radicals aresubstantially as described above.

[0159] Formulations

[0160] Many drugs are inherently hydrophobic and hence have limitedsolubility or ability to be dispersed in an aqueous medium, whichreduces their bioavailability and makes them difficult to formulate oradminister reducing their usefulness. Contrary, other drugs areexcessively hydrophilic and poorly absorbed when given orally.Therefore, certain compounds provided by the present invention arepurposely made hydrophobic. Similarly, a number of potentially usefulbio-active molecules are not sufficiently stable, or have a too shorthalf-life in biological media for successful treatment, which alsolimits their use. As a result of these and other problems ofpharmacokinetics, bioavailability, specificity, etc., there is a need todevelop molecules that can help in the transport or delivery ofbioactive or functional substances.

[0161] Thus, in another aspect, the invention provides formulations ofcompounds of the invention. In addition to the compound of theinvention, the formulations include a second species that interacts withthe compound of the invention to alter a characteristic of the compound,such as its water solubility. In an exemplary embodiment, the compoundof the invention includes a lipid moiety, as described above. The secondspecies includes a lipophilic domain that interacts with the lipidmoiety of the compound of the invention. The second species alsoincludes a hydrophilic moiety that enhances the water solubility of thecomplex formed between the compound of the invention and the secondspecies.

[0162] In an exemplary embodiment, the invention provides a formulationcomprising compound of the invention and a second compound having theformula:

A-B

[0163] wherein A is a hydrophobic domain; and B is a hydrophilic domaincovalently bound to A.

[0164] An exemplary embodiment of the formulations of the invention isset forth in FIG. 2, which is an illustration of the complexes of theinvention formed between the pharmacophore modified with a hydrophobicmodifying group and a poly-ion, such as a polycation. Another exemplaryembodiment is provided by FIG. 3, which is an illustration of thecomplexes of the invention formed between the pharmacophore modifiedwith a hydrophobic modifying group and a dendrimeric poly-ion.

[0165] In a preferred embodiment, the formulations of the invention areaqueous formulations.

[0166] If desired, the formulations may form aggregates. An example ofsuch formulations is constructed of one or more charged lipids inassociation with one or more polymer bearing lipids, optionally inassociation with one or more neutral lipids. The charged lipids mayeither be anionic or cationic. Typically, the lipids are aggregated inthe presence of a multivalent species, such as a counter ion, oppositein charge to the charged lipid. For delivery of prodrugs and/orbioactive agents to selective sites in vivo, aggregates of preferablyunder 2 microns, more preferably under 0.5 microns, and even morepreferably under 200 nm are desired. Most preferably the lipidaggregates are under 200 nm in size and may be as small as 5-10 nm insize.

[0167] When the charged lipid is anionic, a multivalent (divalent,trivalent, etc.) cationic material may be used to form aggregates. It iscontemplated that cations in all of their ordinary valence states willbe suitable for forming aggregates of compounds of the invention.

[0168] When the charged lipid is cationic, an anionic material, forexample, may be used to form aggregates. Preferably, the anionicmaterial is multivalent, such as, for example, divalent. Examples ofuseful anionic materials include monatomic and polyatomic anions such ascarboxylate ions, sulfide ion, sulfite ions, sulfate ions, oxide ions,nitride ions, carbonate ions, and phosphate ions. Anions of ethylenediamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid(DTPA), and 1,4,7,10-tetraazocyclododecane-N′,N′,N″,N″-tetraacetic acid(DOTA) may also be used. Further examples of useful anionic materialsinclude anions of polymers and copolymers of acrylic acid, methacrylicacid, other polyacrylates and methacrylates, polymers with pendant SO₃Hgroups, such as sulfonated polystyrene, and polystyrenes containingcarboxylic acid groups.

[0169] In an exemplary embodiment, the composition of the invention ischarged and a polyion, e.g. a charged dendrimer is used to form anaggregate. Dendrimers are polymers of spherical or otherthree-dimensional shapes that have precisely defined compositions andthat possess a precisely defined molecular weight. Dendrimers can besynthesized as water-soluble macromolecules through appropriateselection of internal and external moieties. See, U.S. Pat. Nos.4,507,466 and 4,568,737, incorporated by reference herein. The firstwell-defined, symmetrical, dendrimer family was the polyamidoamine(PAMAM) dendrimers, which are manufactured by the Dow Chemical Company.Since the synthesis and characterization of the first dendrimers, alarge array of dendrimers of diverse sizes and compositions has beenprepared. See, for example, Liu M. and Frechet J. M. J., Pharm. Sci.Tech. Today 2(11): 393 (1999).

[0170] Dendritic macromolecules are characterized by a highly branched,layered structure with a multitude of chain ends. Dendrimers areparticularly well defined with a very regular and almost sizemonodisperse structure, while hyperbranched polymers are less welldefined and have a broader polydispersity. Dendritic macromolecules areusually constructed from AB_(x) monomers. Hyperbranched polymers aregenerally obtained via a polymerization reaction that generally takesplace in a single series of propagation steps. Dendrimers are generallyobtained by multistep iterative syntheses using either a divergent(Tomalia et al., U.S. Pat. Nos. 4,435,548; 4,507,466, 4,558,120;4,568,737; 5,338,532) or a convergent growth approach (Hawker et al.,U.S. Pat. No. 5,041,516).

[0171] Dendrimers have been conjugated with various pharmaceuticalmaterials as well as with various targeting molecules that may functionto direct the conjugates to selected body locations for diagnostic ortherapeutic applications. See, for example, WO 8801178, incorporated byreference herein. Dendrimers have been used to covalently couplesynthetic porphyrins (e.g., hemes, chlorophyll) to antibody molecules asa means for increasing the specific activity of radiolabeled antibodiesfor tumor therapy and diagnosis. Roberts et al., Bioconjug. Chemistry1:305-308 (1990); Tomalia et al., U.S. Pat. No. 5,714,166.

[0172] Exemplary dendrimers of use in this aspect of the inventioninclude the well-known PAMAM poly(amidoamine) dendrimers or ASTRAMOLpoly(propyleneimine), in part as a result of their easy transformationinto ionically charged species.

[0173] In an exemplary embodiment, the hydrophilic domain of componentB, includes a hydrophilic oligomer or polymer. Suitable hydrophilicgroups include, for example, polyalkyleneoxides such as, for example,polyethylene glycol (PEG) and polypropylene glycol (PPG),polyvinylpyrrolidones, polyvinylmethylethers, polyacrylamides, such as,for example, polymethacrylamides, polydimethylacrylamides andpolyhydroxypropylmethacrylamides, polyhydroxyethyl acrylates,polyhydroxypropyl methacrylates, polymethyloxazolines,polyethyloxazolines, polyhydroxyethyloxazolines,polyhyhydroxypropyloxazolines, polyvinyl alcohols, polyphosphazenes,poly(hydroxyalkylcarboxylic acids), polyoxazolidines, polyaspartamide,and polymers of sialic acid (polysialics). The hydrophilic polymers arepreferably selected from the group consisting of PEG, PPG,polyvinylalcohol and polyvinylpyrrolidone and copolymers thereof, withPEG and PPG polymers being more preferred and PEG polymers being evenmore preferred.

[0174] In another exemplary embodiment, the compound of the inventionhas an oral bioavailability of at least 15%, more preferably at least20% of the administered dose. An exemplary formulation of a compound ofthe invention that provides the desired oral bioavailability is an acidaddition salt of the heterocyclic compound of the invention. The acidaddition salt may be either a salt of a mineral or organic acid, e.g., acarboxylic acid.

[0175] In accordance with the above embodiment, the inventors havesurprisingly discovered that carboxylic acid salts of the compounds ofthe invention provide the desired oral bioavailability. As shown inTable 2, the oral bioavailability of an exemplary carboxylic acid saltof DHAdC is approximately 23%, which is more than twice the oralbioavailability of the corresponding base. TABLE 2^(α) DHAdC- DHAdC-DHAdC- base (9) base (9) palmitate (27) (IV) (Oral) Oral Cmax (ng/ml)40,034 1,858 2,816 AUC_(∞)(ng-hr/ml) 74,975 8,538 17,552 Half-life (hr)1.8 0.8 0.7 % oral 11 23 bioavailability

[0176] Carrier Molecules

[0177] The compounds of the invention and their formulations can alsoinclude a carrier molecule, useful to target the pharmacophore to aspecific region within the body or tissue, or to a selected species orstructure in vitro. Selective targeting of an agent by its attachment toa species with an affinity for the targeted region is well known in theart. Both small molecule and polymeric targeting agents are of use inthe present invention.

[0178] In an exemplary embodiment, a compound of the invention is linkedto a targeting agent that selectively delivers it to a cell, organ orregion of the body. Exemplary targeting agents such as antibodies,ligands for receptors, lectins, saccharides, antibodies, and the likeare recognized in the art and are useful without limitation inpracticing the present invention. Other targeting agents include a classof compounds that do not include specific molecular recognition motifsinclude macromolecules such as poly(ethylene glycol), polysaccharide,polyamino acids and the like, which add molecular mass to the ligand.The ligand-targeting agent conjugates of the invention are exemplifiedby the use of a nucleic acid-ligand conjugate. The focus onligand-oligonucleotide conjugates is for clarity of illustration and isnot limiting of the scope of targeting agents to which the ligands (orcomplexes) of the invention can be conjugated. Moreover, it isunderstood that “ligand” refers to both the free ligand and its metalcomplexes.

[0179] Exemplary nucleic acid targeting agents include aptamers,antisense compounds, and nucleic acids that form triple helices.Typically, a hydroxyl group of a sugar residue, an amino group from abase residue, or a phosphate oxygen of the nucleotide is utilized as theneeded chemical functionality to couple the nucleotide-based targetingagent to the ligand. However, one of skill in the art will readilyappreciate that other “non-natural” reactive functionalities can beappended to a nucleic acid by conventional techniques. For example, thehydroxyl group of the sugar residue can be converted to a mercapto oramino group using techniques well known in the art.

[0180] Aptamers (or nucleic acid antibody) are single- ordouble-stranded DNA or single-stranded RNA molecules that bind specificmolecular targets. Generally, aptamers function by inhibiting theactions of the molecular target, e.g., proteins, by binding to the poolof the target circulating in the blood. Aptamers possess chemicalfunctionality and thus, can covalently bond to ligands, as describedherein.

[0181] Although a wide variety of molecular targets are capable offorming non-covalent but specific associations with aptamers, includingsmall molecules drugs, metabolites, cofactors, toxins, saccharide-baseddrugs, nucleotide-based drugs, glycoproteins, and the like, generallythe molecular target will comprise a protein or peptide, including serumproteins, kinins, eicosanoids, cell surface molecules, and the like.Examples of aptamers include Gilead's antithrombin inhibitor GS 522 andits derivatives (Gilead Science, Foster City, Calif.). See also, Macayaet al. Proc. Natl. Acad. Sci. USA 90: 3745-9 (1993); Bock et al. Nature(London) 355: 564-566 (1992) and Wang et al. Biochem. 32: 1899-904(1993).

[0182] Aptamers specific for a given biomolecule can be identified usingtechniques known in the art. See, e.g., Toole et al. (1992) PCTPublication No. WO 92/14843; Tuerk and Gold (1991) PCT Publication No.WO 91/19813; Weintraub and Hutchinson (1992) PCT Publication No.92/05285; and Ellington and Szostak, Nature 346: 818 (1990). Briefly,these techniques typically involve the complexation of the moleculartarget with a random mixture of oligonucleotides. The aptamer-moleculartarget complex is separated from the uncomplexed oligonucleotides. Theaptamer is recovered from the separated complex and amplified. Thiscycle is repeated to identify those aptamer sequences with the highestaffinity for the molecular target.

[0183] For diseases that result from the inappropriate expression ofgenes, specific prevention or reduction of the expression of such genesrepresents an ideal therapy. In principle, production of a particulargene product may be inhibited, reduced or shut off by hybridization of asingle-stranded deoxynucleotide or ribodeoxynucleotide complementary toan accessible sequence in the mRNA, or a sequence within the transcriptthat is essential for pre-mRNA processing, or to a sequence within thegene itself. This paradigm for genetic control is often referred to asantisense or antigene inhibition. Additional efficacy is imparted vy theconjugation to the nucleic acid of an alkylating agent, such as those ofthe present invention.

[0184] Antisense compounds are nucleic acids designed to bind anddisable or prevent the production of the mRNA responsible for generatinga particular protein. Antisense compounds include antisense RNA or DNA,single or double stranded, oligonucleotides, or their analogs, which canhybridize specifically to individual mRNA species and preventtranscription and/or RNA processing of the mRNA species and/ortranslation of the encoded polypeptide and thereby effect a reduction inthe amount of the respective encoded polypeptide. Ching et al. Proc.Natl. Acad. Sci. U.S.A. 86: 10006-10010 (1989); Broder et al. Ann. Int.Med. 113: 604-618 (1990); Loreau et al. FEBS Letters 274: 53-56 (1990);Holcenberg et al. WO91/11535; WO91/09865; WO91/04753; WO90/13641; WO91/13080, WO 91/06629, and EP 386563). Due to their exquisite targetsensitivity and selectivity, antisense oligonucleotides are useful fordelivering therapeutic agents, such as the ligands of the invention to adesired molecular target.

[0185] The site specificity of nucleic acids (e.g., antisense compoundsand triple helix drugs) is not significantly affected by modification ofthe phosphodiester linkage or by chemical modification of theoligonucleotide terminus. Consequently, these nucleic acids can bechemically modified; enhancing the overall binding stability, increasingthe stability with respect to chemical degradation, increasing the rateat which the oligonucleotides are transported into cells, and conferringchemical reactivity to the molecules. The general approach toconstructing various nucleic acids useful in antisense therapy has beenreviewed by van der Krol et al., Biotechniques 6: 958-976 (1988) andStein et al. Cancer Res. 48: 2659-2668 (1988). Therefore, in anexemplary embodiment, the ligands of the invention are conjugated to anucleic acid by modification of the phosphodiester linkage.

[0186] Moreover, aptamers, antisense compounds and triple helix drugsbearing compounds of the invention can also can include nucleotidesubstitutions, additions, deletions, or transpositions, so long asspecific hybridization to or association with the relevant targetsequence is retained as a functional property of the oligonucleotide.For example, some embodiments will employ phosphorothioate analogs whichare more resistant to degradation by nucleases than their naturallyoccurring phosphate diester counterparts and are thus expected to have ahigher persistence in vivo and greater potency (see, e.g., Campbell etal., J. Biochem. Biophys. Methods 20: 259-267(1990)). Phosphoramidatederivatives of oligonucleotides also are known to bind to complementarypolynucleotides and have the additional capability of accommodatingcovalently attached ligand species and will be amenable to the methodsof the present invention. See, for example, Froehier et al., NucleicAcids Res. 16(11): 4831 (1988).

[0187] Terminal modification also provides a useful procedure toconjugate the pharmacophore to the nucleic acid, modify cell typespecificity, pharmacokinetics, nuclear permeability, and absolute celluptake rate for oligonucleotide pharmaceutical agents. For example, anarray of substitutions at the 5′ and 3′ ends to include reactive groupsare known, which allow covalent attachment of the cytotoxins. See, e.g.,OLIGODEOXYNUCLEOTIDES: ANTISENSE INHIBITORS OF GENE EXPRESSION, (1989)Cohen, Ed., CRC Press; PROSPECTS FOR ANTISENSE NUCLEIC ACID THERAPEUTICSFOR CANCER AND AIDS, (1991), Wickstrom, Ed., Wiley-Liss; GENEREGULATION: BIOLOGY OF ANTISENSE RNA AND DNA, (1992) Erickson and Izant,Eds., Raven Press; and ANTISENSE RNA AND DNA, (1992), Murray, Ed.,Wiley-Liss. For general methods relating to antisense compounds, see,ANTISENSE RNA AND DNA, (1988), D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

[0188] In another exemplary embodiment, the invention utilizes apeptide-based targeting moiety. Generally speaking, peptides that areparticularly useful as targeting ligands include natural, modifiednatural, or synthetic peptides that incorporate additional modes ofresistance to degradation by vascularly circulating esterases, amidases,or peptidases. Suitable targeting ligands, and methods for theirpreparation, will be readily apparent to one skilled in the art, in viewof the disclosure herein. Exemplary targeting ligands in the presentinvention include cell adhesion molecules (CAM), among which are, forexample, cytokines, integrins, cadherins, immunoglobulins and selectins.

[0189] Regarding targeting to specific cell types, for example,endothelial cells, suitable targeting ligands include, for example, oneor more of the following: growth factors, including, for example, basicfibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), platelet-derived endothelial cell growth factor(PD-ECGF) vascular endothelial growth factor (VEGF) and human growthfactor (HGF); angiogenin; tumor necrosis factors, including tumornecrosis factor-α (TNF-α) and tumor necrosis factor-β (TNF-β), andreceptor antibodies and fragments thereof to tumor necrosis factor (TNF)receptor 1 or 2 family, including, for example, TNF-R1, TNF-R2, FAS,TNFR-RP, NGF-R, CD30, CD40, CD27, OX40 and 4-1BB; copper-containingpolyribonucleotide angiotropin with a molecular weight of about 4,500,as well as low molecular weight non-peptide angiogenic factors, such as1-butyryl glycerol; the prostaglandins, including, for example,prostaglandin E₁ (PGE₁) and prostaglandin E₂ (PGE₂); nicotinamide;adenosine; dipyridamole; dobutamine; hyaluronic acid degradationproducts, such as, for example, degradation products resulting fromhydrolysis of β-linkages, including hyalobiuronic acid; angiogenesisinhibitors, including, for example, collagenase inhibitors; minocycline;medroxyprogesterone; chitin chemically modified with 6-O-sulfate and6-O-carboxymethyl groups; angiostatic steroids, such astetrahydrocortisol; and heparin, including fragments of heparin, suchas, for example, fragments having a molecular weight of about 6,000,admixed with steroids, such as, for example, cortisone orhydrocortisone; angiogenesis inhibitors, including angioinhibin(AGM-1470- an angiostatic antibiotic); platelet factor 4; protamine;sulfated polysaccharide peptidoglycan complexes derived from thebacterial wall of an Arthobacter species; fungal-derived angiogenesisinhibitors, such as fumagillin derived from Aspergillus fumigatus;D-penicillamine; gold thiomalate; thrombospondin; vitamin D₃ analogues;interferons, including, for example, α-interferon, β-interferon andγ-interferon; cytokines and cytokine fragments, such as theinterleukins, including, for example, interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-5 (IL-5) andinterleukin-8 (IL-8); erythropoietin; a 20-mer peptide or smaller forbinding to receptor or antagonists to native cytokines; granulocytemacrophage colony stimulating factor (GMCSF); LTB₄ leukocyte receptorantagonists; heparin, including low molecular weight fragments ofheparin or analogues of heparin; simple sulfated polysaccharides, suchas cyclodextrins, including α-, β- and γ-cyclodextrin; tetradecasulfate;transferrin; ferritin; platelet factor 4; protamine; Gly-His-Lyscomplexed to copper; ceruloplasmin; (12R)-hydroxyeicosatrienoic acid;okadaic acid; lectins; antibodies; CD11a/CD18; and Very Late ActivationIntegrin-4 (VLA4). Peptides that bind the interluekin-1 (IL-1) receptormay be used.

[0190] The cadherin family of cell adhesion molecules may also be usedas targeting ligands, including for example, the E-, N-, andP-cadherins, cadherin-4, cadherin-5, cadherin-6, cadherin-7, cadherin-8,cadherin-9, cadherin-10, and cadherin-11; and most preferably cadherinC-5. Further, antibodies directed to cadherins, such as, for example,the monoclonal antibody Ec6C10, may be used to recognize cadherinsexpressed locally by specific endothelial cells.

[0191] A wide variety of different targeting ligands can be selected tobind to the cytoplasmic domains of the ELAM molecules. Targeting ligandsin this regard may include lectins, a wide variety of carbohydrate orsugar moieties, antibodies, antibody fragments, Fab fragments, such as,for example, Fab′2, and synthetic peptides, including, for example,Arginine-Glycine-Aspartic Acid (R-G-D) which may be targeted to woundhealing. While many of these materials may be derived from naturalsources, some may be synthesized by molecular biological recombinanttechniques and others may be synthetic in origin. Peptides may beprepared by a variety of different combinatorial chemistry techniques asare now known in the art. Targeting ligands derived or modified fromhuman leukocyte origin, such as CD11a/CD18, and leukocyte cell surfaceglycoprotein (LFA-1), may also be used as these are known to bind to theendothelial cell receptor ICAM-1. The cytokine inducible member of theimmunoglobulin superfamily, VCAM-1, which is mononuclearleukocyte-selective, may also be used as a targeting ligand. VLA4,derived from human monocytes, may be used to target VCAM-1.

[0192] As with the endothelial cells discussed above, a wide variety ofpeptides, proteins and antibodies may be employed as targeting ligandsfor targeting epithelial cells. Preferably, a peptide, includingsynthetic, semi-synthetic or naturally-occurring peptides, with highaffinity to the epithelial cell target receptor may be selected, withsynthetic peptides being more preferred. In connection with thesepreferred embodiments, peptides having from about 5 to about 15 aminoacid residues are preferred. Antibodies may be used as whole antibody orantibody fragments, for example, Fab or Fab′2, either of natural orrecombinant origin. The antibodies of natural origin may be of animal orhuman origin, or may be chimeric (mouse/human). Human recombinant orchimeric antibodies are preferred and fragments are preferred to wholeantibody.

[0193] In one embodiment of the invention, the targeting ligands aredirected toward lymphocytes which may be T-cells or B-cells, withT-cells being the preferred target. To select a class of targetedlymphocytes, a targeting ligand having specific affinity for that classis employed. For example, an anti CD4 antibody can be used for selectingthe class of T-cells harboring CD4 receptors, an anti CD-8 antibody canbe used for selecting the class of T-cells harboring CD-8 receptors, ananti CD-34 antibody can be used for selecting the class of T-cellsharboring CD-34 receptors, etc. A lower molecular weight ligand ispreferably employed, e.g., Fab or a peptide fragment. For example, anOKT3 antibody or OKT3 antibody fragment may be used.

[0194] When a receptor for a class of T-cells or clones of T-cells isselected, the steroid prodrug will be delivered to that class of cells.Using HLA-derived peptides, for example, will allow selection oftargeted clones of cells expressing reactivity to HLA proteins.

[0195] Another useful area for targeted prodrug delivery involves theinterleukin-2 (IL-2) system. IL-2 is a t-cell growth factor producedfollowing antigen or mitogen induced stimulation of lymphoid cells.Among the cell types producing IL-2 are CD4⁺ and CD8^(t)-cells and largegranular lymphocytes, as well as certain t-cell tumors. IL-2 receptorsare glycoproteins expressed on responsive cells. They are notable inconnection with the present invention because they are readilyendocytosed into lysosomal inclusions when bound to IL-2. The ultimateeffect of this endocytosis depends on the target cell, but among thenotable in vivo effects are regression of transplantable murine tumors,human melanoma or renal cell cancer. IL-2 has also been implicated inantibacterial and antiviral therapies and plays a role in allograftrejection. In addition to IL-2 receptors, preferred targets include theanti-IL-2 receptor antibody, natural IL-2 and an IL-2 fragment of a20-mer peptide or smaller generated by phage display that binds to theIL-2 receptor.

[0196] Although not intending to be bound by any particular theory ofoperation, IL-2 can be conjugated to the prodrugs and/or other deliveryvehicles and thus mediate the targeting of cells bearing IL-2 receptors.Endocytosis of the ligand-receptor complex would then deliver thesteroid to the targeted cell, thereby inducing its death throughapoptosis—independent and superceding any proliferative or activatingeffect that IL-2 would promote alone.

[0197] Additionally, an IL-2 peptide fragment which has binding affinityfor IL-2 receptors can be incorporated either by direct attachment to areactive moiety on the steroid prodrug or via a spacer or linkermolecule with a reactive end such as an amine, hydroxyl, or carboxylicacid functional group. Such linkers are well known in the art and maycomprise from 3 to 20 amino acid residues. Alternatively, D-amino acidsor derivatized amino acids may be used which avoid proteolysis in thetarget tissue.

[0198] Still other systems which can be used in the present inventioninclude IgM-mediated endocytosis in B-cells or a variant of theligand-receptor interactions described above wherein the T-cell receptoris CD2 and the ligand is lymphocyte function-associated antigen 3(LFA-3), as described, for example, by Wallner et al, J ExperimentalMed., 166: 923-932 (1987), the disclosure of which is herebyincorporated by reference herein in its entirety.

[0199] The targeting ligand may be incorporated in the presentstabilizing materials in a variety of ways. Generally speaking, thetargeting ligand may be incorporated in the present stabilizingmaterials by being associated covalently or non-covalently with one ormore of the stabilizing materials which are included in the compositionsincluding, for example, the prodrugs, lipids, proteins, polymers,surfactants, and/or auxiliary stabilizing materials. In preferred form,the targeting ligand may be associated covalently with one or more ofthe aforementioned materials contained in the present stabilizingmaterials. Preferred stabilizing materials of the present inventioncomprise prodrugs, lipid, protein, polymer or surfactant compounds. Inthese compositions, the targeting ligands are preferably associatedcovalently with the prodrug, lipid, protein, polymer or surfactantcompounds.

[0200] The covalent linking of the targeting ligands to thepharmacophores in the present compositions, including the prodrugs, andlipid components is accomplished using synthetic organic techniqueswhich are readily apparent to one of ordinary skill in the art in viewof the present disclosure. For example, the targeting ligands may belinked to the materials, including the lipids, via the use of well-knowncoupling or activation agents. As known to the skilled artisan,activating agents are generally electrophilic, which can be employed toelicit the formation of a covalent bond. Exemplary activating agentsinclude, for example, carbonyldiimidazole (CDI),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), methylsulfonyl chloride, Castro's Reagent, and diphenyl phosphoryl chloride.

[0201] The covalent bonds optionally involve crosslinking and/orpolymerization. Crosslinking preferably refers to the attachment of twochains of polymer molecules by bridges, composed of an element, a group,or a compound, which join certain carbon atoms of the chains by covalentchemical bonds. For example, crosslinking may occur in polypeptides thatare joined by the disulfide bonds of the cystine residue. Crosslinkingmay be achieved, for example, by (1) adding a chemical substance(crosslinking agent) and exposing the mixture to heat, or (2) subjectinga polymer to high-energy radiation. A variety of crosslinking agents, or“tethers”, of different lengths and/or functionalities are described,for example, in R. L. Lunbland, Techniques in Protein Modification, CRCPress, Inc., Ann Arbor, Mich., pp. 249-68 (1995), the disclosures ofwhich is hereby incorporated herein by reference in its entirety.Exemplary crosslinkers include, for example,3,3′-dithiobis(succinimidylpropionate), dimethyl suberimidate, and itsvariations thereof, based on hydrocarbon length, andbis-N-maleimido-1,8-octane.

[0202] Standard peptide methodology may be used to link the targetingligand to the compound of the invention utilizing linker groups havingtwo unique terminal functional groups. Bifunctional hydrophilicpolymers, and especially bifunctional PEGs, may be synthesized usingstandard organic synthetic methodologies. In addition, many of thesematerials are available commercially, such as, for example,α-amino-ω-carboxy-PEG that is commercially available from ShearwaterPolymers (Huntsville, Ala.). An advantage of using a PEG material as thelinking group is that the size of the PEG can be varied such that thenumber of monomeric subunits of ethylene glycol may be as few as, forexample, about 5, or as many as, for example, about 500 or even greater.Accordingly, the “tether” or length of the linkage may be varied, asdesired. This may be important depending, for example, on the particulartargeting ligand employed.

[0203] In an exemplary embodiment, the terminus of the hydrophilicspacer, such as polyethylene glycol ethylamine, which contains areactive group, such as an amine or hydroxyl group, is used to bind atargeting ligand to a compound of the invention. For example,polyethylene glycol ethylamine may be reacted with N-succinimidylbiotinor p-nitrophenylbiotin to introduce onto the spacer a useful couplinggroup.

[0204] The carrier molecules may also be used as a backbone forcompounds of the invention that are poly- or multi-valent species,including, for example, species such as dimers, trimers, tetramers andhigher homologs of the compounds of the invention. The poly- andmulti-valent species can be assembled from a single species or more thanone species of the invention. For example, a dimeric construct can be“homo-dimeric” or “heterodimeric.” Moreover, poly- and multi-valentconstructs in which a compound of the invention, or a reactive analoguethereof, is attached to an oligomeric or polymeric framework (e.g.,polylysine, dextran, hydroxyethyl starch and the like) are within thescope of the present invention. The framework is preferablypolyfunctional (i.e. having an array of reactive sites for attachingcompounds of the invention). Moreover, the framework can be derivatizedwith a single species of the invention or more than one species of theinvention.

[0205] Moreover, the properties of the carrier molecule can be selectedto afford compounds having water-solubility that is enhanced relative toanalogous compounds that are not similarly functionalized. Thus, any ofthe substituents set forth herein can be replaced with analogousradicals that have enhanced water solubility. For example, it is withinthe scope of the invention to, for example, replace a hydroxyl groupwith a diol, or an amine with a quaternary amine, hydroxylamine orsimilar more water-soluble moiety. In a preferred embodiment, additionalwater solubility is imparted by substitution at a site not essential forthe activity towards the ion channel of the compounds set forth hereinwith a moiety that enhances the water solubility of the parentcompounds. Methods of enhancing the water-solubility of organiccompounds are known in the art. Such methods include, but are notlimited to, functionalizing an organic nucleus with a permanentlycharged moiety, e.g., quaternary ammonium, or a group that is charged ata physiologically relevant pH, e.g. carboxylic acid, amine. Othermethods include, appending to the organic nucleus hydroxyl- oramine-containing groups, e.g. alcohols, polyols, polyethers, and thelike. Representative examples include, but are not limited to,polylysine, polyethyleneimine, poly(ethyleneglycol) andpoly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art. See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991.

[0206] Pharmaceutical Formulations

[0207] In another preferred embodiment, the present invention provides apharmaceutical formulation comprising a dendrimer-agent conjugate and apharmaceutically acceptable carrier.

[0208] The compounds described herein, or pharmaceutically acceptableaddition salts or hydrates thereof, can be delivered to a patient usinga wide variety of routes or modes of administration. Suitable routes ofadministration include, but are not limited to, inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.

[0209] The compounds described herein, or pharmaceutically acceptablesalts, and/or hydrates thereof, may be administered singly, incombination with other compounds of the invention, and/or in cocktailscombined with other therapeutic agents. Of course, the choice oftherapeutic agents that can be co-administered with the compounds of theinvention will depend, in part, on the condition being treated.

[0210] For example, when administered to a patient undergoing cancertreatment, the compounds may be administered in cocktails containingother bioactive agents, such as anti-cancer agents and/or supplementarypotentiating agents. The compounds may also be administered in cocktailscontaining agents that treat the side-effects of radiation therapy, suchas anti-emetics, radiation protectants, etc.

[0211] Other suitable bioactive agents include, for example,antineoplastic agents, such as platinum compounds (e.g., spiroplatin,cisplatin, and carboplatin), methotrexate, adriamycin, taxol, mitomycin,ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine,mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan(e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine, mitotane,procarbazine hydrochloride dactinomycin (actinomycin D), daunorubicinhydrochloride, doxorubicin hydrochloride, mitomycin, plicamycin(mithramycin), aminoglutethimide, estramustine phosphate sodium,flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate,testolactone, trilostane, amsacrine (m-AMSA), asparaginase(L-asparaginase) Erwina asparaginase, etoposide (VP-16), interferonα-2a, interferon α-2b, teniposide (VM-26), vinblastine sulfate (VLB),vincristine sulfate, bleomycin, bleomycin sulfate, methotrexate,adriamycin, and arabinosyl; blood products such as parenteral iron,hemin, hematoporphyrins and their derivatives; biological responsemodifiers such as muramyldipeptide, muramyltripeptide, microbial cellwall components, lymphokines (e.g., bacterial endotoxin such aslipopoly-saccharide, macrophage activation factor), sub-units ofbacteria (such as Mycobacteria and Corynebacteria), the syntheticdipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine; antifungal agentssuch as ketoconazole, nystatin, griseofulvin, flucytosine (5-fc),miconazole, amphotericin B, ricin, and β-lactam antibiotics (e.g.,sulfazecin); hormones and steroids such as growth hormone, melanocytestimulating hormone, estradiol, beclomethasone dipropionate,betamethasone, betamethasone acetate and betamethasone sodium phosphate,vetamethasone disodium phosphate, vetamethasone sodium phosphate,cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasonesodium phosphate, flunsolide, hydrocortisone, hydrocortisone acetate,hydrocortisone cypionate, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, methylprednisolone, methylprednisoloneacetate, methylprednisolone sodium succinate, paramethasone acetate,prednisolone, prednisolone acetate, prednisolone sodium phosphate,prednisolone tebutate, prednisone, triamcinolone, triamcinoloneacetonide, triamcinolone diacetate, triamcinolone hexacetonide andfludrocortisone acetate; vitamins such as cyanocobalamin neinoic acid,retinoids and derivatives such as retinol palmitate, and α-tocopherol;peptides, such as manganese super oxide dimutase; enzymes such asalkaline phosphatase; anti-allergic agents such as amelexanox;anti-coagulation agents such as phenprocoumon and heparin; circulatorydrugs such as propranolol; metabolic potentiators such as glutathione;antituberculars such as para-aminosalicylic acid, isoniazid, capreomycinsulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,rifampin, and streptomycin sulfate; antivirals such as acyclovir,amantadine azidothymidine (AZT or Zidovudine), ribavirin, amantadine,vidarabine, and vidarabine monohydrate (adenine arabinoside, ara-A);antianginals such as diltiazem, nifedipine, verapamil, erythrityltetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate)and pentaerythritol tetranitrate; anticoagulants such as phenprocoumon,heparin; antibiotics such as dapsone, chloramphenicol, neomycin,cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin,lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,nafcillin, oxacillin, penicillin Gm penicillin V, ticarcillin rifampinand tetracycline; antiinflammatories such as diffinisal, ibuprofen,indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates;antiprotozoans such as chloroquine, hydroxychloroquine, metronidazole,quinine and meglumine antimonate; antirheumatics such as penicillamine;narcotics such as paregoric; opiates such as codeine, heroin, methadone,morphine and opium; cardiac glycosides such as deslanoside, digitoxin,digoxin, digitalin and digitalis; neuromuscular blockers such asatracurium besylate, gallamine triethiodide, hexafluorenium bromide,metocurine iodide, pancuronium bromide, succinylcholine chloride(suxamethonium chloride), tubocurarine chloride and vecuronium bromide;sedatives (hypnotics) such as amobarbital, amobarbital sodium,aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol,ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazinehydrochloride, methyprylon, midazolam hydrochloride, paraldehyde,pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbitalsodium, talbutal, temazepam and triazolam; local anesthetics such asbupivacaine hydrochloride, chloroprocaine hydrochloride, etidocainehydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride,procaine hydrochloride and tetracaine hydrochloride; general anestheticssuch as droperidol, etomidate, fentanyl citrate with droperidol,ketamine hydrochloride, methohexital sodium and thiopental sodium; andradioactive particles or ions such as strontium, iodide rhenium andyttrium. In certain referred embodiments, the bioactive agent is amonoclonal antibody, such as a monoclonal antibody capable of binding tomelanoma antigen.

[0212] The active compound(s) of the invention are administered per seor in the form of a pharmaceutical composition wherein the activecompound(s) is in admixture with one or more pharmaceutically acceptablecarriers, excipients or diluents. Pharmaceutical compositions for use inaccordance with the present invention are typically formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries, which facilitateprocessing of the active compounds into preparations which, can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

[0213] For injection, the agents of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0214] For oral administration, the compounds can be formulated readilyby combining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by combining the dendrimer with a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, for example,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

[0215] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,poly(ethylene oxide), and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0216] Pharmaceutical preparations, which can be used orally, includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0217] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0218] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0219] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof such as sodium alginate.

[0220] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions.

[0221] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0222] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0223] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation ortranscutaneous delivery (e.g., subcutaneously or intramuscularly),intramuscular injection or a transdermal patch. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

[0224] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as poly(ethylene oxide).

[0225] Microarrays

[0226] The invention also provides microarrays including immobilizedcompounds of the invention and compounds functionalized with compoundsof the invention. Moreover, the invention provides methods ofinterrogating microarrays using probes that are functionalized withcompounds of the invention. The immobilized species and the probes areselected from substantially any type of molecule, including, but notlimited to, small molecules, peptides, enzymes nucleic acids and thelike.

[0227] Nucleic acid microarrays consisting of a multitude of immobilizednucleic acids are revolutionary tools for the generation of genomicinformation, see, Debouck et al., in supplement to Nature Genetics,21:48-50 (1999). The discussion that follows focuses on the use ofcompounds of the invention in conjunction with nucleic acid microarrays.This focus is intended to be illustrative and does not limit the scopeof materials with which this aspect of the present invention can bepracticed.

[0228] Thus, in another preferred embodiment, the compounds of thepresent invention are utilized in a microarray format. The compound ofthe invention, or species bearing a compound of the invention canthemselves be components of a microarray or, alternatively they can beutilized as a tool to screen components of a microarray.

[0229] In an exemplary embodiment, the microarrays comprise n probesthat comprise identical or different nucleic acid sequences.Alternatively, the microarray can comprise a mixture of n probescomprising groups of identical and different nucleic acid sequencesidentical nucleic acid sequences). In a preferred embodiment, n is anumber from 2 to 100, more preferably, from 10 to 1,000, and morepreferably from 100 to 10,000. In a still further preferred embodiment,the n probes are patterned on a substrate as n distinct locations in amanner that allows the identity of each of the n locations to beascertained.

[0230] In yet another preferred embodiment, the invention also providesa method for preparing a microarray of n probes. The method includesattaching the probes to selected regions of a substrate. A variety ofmethods are currently available for making arrays of biologicalmacromolecules, such as arrays nucleic acid molecules.

[0231] One method for making ordered arrays of probes on a substrate isa “dot blot” approach. In this method, a vacuum manifold transfers aplurality, e.g., 96, aqueous samples of probes from 3 millimeterdiameter wells to a substrate. The probe is immobilized on the porousmembrane by baking the membrane or exposing it to UV radiation. A commonvariant of this procedure is a “slot-blot” method in which the wellshave highly-elongated oval shapes.

[0232] Another technique employed for making ordered arrays of probesuses an array of pins dipped into the wells, e.g., the 96 wells of amicrotiter plate, for transferring an array of samples to a substrate,such as a porous membrane. One array includes pins that are designed tospot a membrane in a staggered fashion, for creating an array of 9216spots in a 22×22 cm area. See, Lehrach, et al., HYBRIDIZATIONFINGERPRINTING IN GENOME MAPPING AND SEQUENCING, GENOME ANALYSIS, Vol.1, Davies et al, Eds., Cold Springs Harbor Press, pp. 39-81 (1990).

[0233] An alternate method of creating ordered arrays of probes isanalogous to that described by Pirrung et al. (U.S. Pat. No. 5,143,854,issued 1992), and also by Fodor et al., (Science, 251: 767-773 (1991)).This method involves synthesizing different probes at different discreteregions of a particle or other substrate. This method is preferably usedwith relatively short probe molecules, e.g., less than 20 bases. Arelated method has been described by Southern et al. (Genomics, 13:1008-1017 (1992)).

[0234] Khrapko, et al., DNA Sequence, 1: 375-388 (1991) describes amethod of making an nucleic acid matrix by spotting DNA onto a thinlayer of polyacrylamide. The spotting is done manually with amicropipette.

[0235] The substrate can also be patterned using techniques such asphotolithography (Kleinfield et al., J. Neurosci. 8:4098-120 (1998)),photoetching, chemical etching and microcontact printing (Kumar et al.,Langmuir 10: 1498-511 (1994)). Other techniques for forming patterns ona substrate will be readily apparent to those of skill in the art.

[0236] The size and complexity of the pattern on the substrate islimited only by the resolution of the technique utilized and the purposefor which the pattern is intended. For example, using microcontactprinting, features as small as 200 nm are layered onto a substrate. See,Xia, Y., J. Am. Chem. Soc. 117:3274-75 (1995). Similarly, usingphotolithography, patterns with features as small as 1 μm are produced.See, Hickman et al., J. Vac. Sci. Technol. 12:607-16 (1994). Patternswhich are useful in the present invention include those which includefeatures such as wells, enclosures, partitions, recesses, inlets,outlets, channels, troughs, diffraction gratings and the like.

[0237] In a presently preferred embodiment, the patterning is used toproduce a substrate having a plurality of adjacent wells, indentationsor holes to contain the probes. In general, each of these substratefeatures is isolated from the other wells by a raised wall or partitionand the wells do not fluidically communicate. Thus, a particle, or othersubstance, placed in a particular well remains substantially confined tothat well. In another preferred embodiment, the patterning allows thecreation of channels through the device whereby an analyte or othersubstance can enter and/or exit the device.

[0238] In another embodiment, the probes are immobilized by “printing”them directly onto a substrate or, alternatively, a “lift off” techniquecan be utilized. In the lift off technique, a patterned resist is laidonto the substrate, an organic layer is laid down in those areas notcovered by the resist and the resist is subsequently removed. Resistsappropriate for use with the substrates of the present invention areknown to those of skill in the art. See, for example, Kleinfield et al.,J. Neurosci. 8:4098-120 (1998). Following removal of the photoresist, asecond probe, having a structure different from the first probe can bebonded to the substrate on those areas initially covered by the resist.Using this technique, substrates with patterns of probes havingdifferent characteristics can be produced. Similar substrateconfigurations are accessible through microprinting a layer with thedesired characteristics directly onto the substrate. See, Mrkish et al.Ann. Rev. Biophys. Biomol. Struct. 25:55-78 (1996).

[0239] The Methods

[0240] The compounds of the present invention can be used to treat viraldiseases. In addition, the compounds of the present invention can beused to treat cancer and other diseases of deregulated cellularproliferation.

[0241] Without wishing to be bound by theory, for treatment of viraldiseases, the nucleoside and nucleotide analogues of the presentinvention are incorporated into the viral genome. The nucleoside andnucleotide analogues have phosphodiester linkages or acquirephosphodiester linkages, allowing them to be incorporated and extendedby a polymerase. The nucleoside and nucleotide analogues have alteredbase-pairing properties allowing incorporation of mutations into theviral genome, dramatically increasing the viral mutation rate. Theincrease in viral mutation rate results in decreased viability ofprogeny virus, thereby inhibiting viral replication. In presentlypreferred embodiments, 5-aza-2′-deoxycytidine, 5-aza-cytidine, andderivatives and variants thereof are used to treat DNA viruses, RNAviruses, and retrovirus infections.

[0242] The compounds of the present invention can also be used to treatcancer. Without wishing to be bound by theory, the nucleoside andnucleotide analogues of the present invention are incorporated into thenucleic acids of a cancerous cell, either DNA or RNA. The nucleoside andnucleotide analogues have phosphodiester linkages or acquirephosphodiester linkages, allowing them to be incorporated and extendedby a polymerase. In one embodiment, the nucleoside and nucleotideanalogues have altered base-pairing properties allowing incorporation ofmutations into the genome of the cancer cell, dramatically increasingthe mutation rate in the cancer cell. The increased mutation rateresults in decreased viability of progeny cells, leading to death of thecancer cells, or a diminished growth rate, or inability to metastasize.In another embodiment, mutations are incorporated into transcriptionproducts, e.g., mRNA molecules that encode proteins or tRNA moleculesuseful for translation of proteins. The mutated transcription productsencode mutated proteins, for example, proteins with altered amino acidsequences or truncations that lead, in turn to the inactivation of theprotein. The inability of the cancer cell to consistently encode activeprotein can also result in death of the cancer cells, or a diminishedgrowth rate, or inability to metastasize, or inability to proliferate.

[0243] Assays for Mutagenic Nucleosides and Nucleotides

[0244] In one embodiment, preferred nucleoside analogs of the presentinvention include 5-aza-cytidine, 5-aza-2′-deoxycytidine, andderivatives and variants thereof including nucleotides, which can beincorporated and extended by a polymerase. Generally, such analogs havephosphodiester linkages allowing them to be extended by the polymerasemolecule after their incorporation into RNA or DNA. Thus, unlike certainviral inhibitors which cause chain termination (e.g., analogs lacking a3′-hydroxyl group), the preferred analogs of the present invention arenon-chain-terminating analogs that generally do not result in thetermination of RNA or DNA synthesis upon their incorporation. Instead,they are preferably error-inducing analogs, which can be incorporatedinto an DNA or RNA product but which effectively alter the base-pairingproperties at the position of their incorporation, thereby causing theintroduction of errors in the RNA or DNA sequence at the site ofincorporation.

[0245] Determination of parameters concerning the incorporation ofaltered nucleotides by a polymerase such as, human RNA polymerase II andviral polymerases/replicates or the phosphorylation of nucleosideanalogs by cellular kinase, is made by methods analogous to those usedfor incorporation of deoxynucleoside triphosphates by DNA polymerases(Boosalis, et al., J. Biol. Chem. 262: 14689-14698 (1987). Those ofskill in the art will recognize that such assays can also be used todetermine the ability of a compound to inhibit a cellular polymerase orto determine the replicative capability of a virus that has been treatedwith an altered nucleotide. In selected situations direct determinationof the frequency of mutations that are introduced into the viral genome(Ji and Loeb, Virol., 199: 323-330 (1994) can be made.

[0246] The nucleoside or nucleotide analog is incorporated by a cellularpolymerase or viral polymerase into the DNA or RNA copy of the genomicnucleic acid with an efficiency of at least about 0.1%, preferably atleast about 5%, and most preferably equal to that of a naturallyoccurring complementary nucleic acid when compared in equal amounts inan in vitro assay. Thus, an error rate of about 1 in 1000 bases or morewould be sufficient to enhance mutagenesis of the virus. The ability ofthe nucleoside or nucleotide analog to cause incorrect base pairing maybe determined by testing and examining the frequency and nature ofmutations produced by the incorporation of an analog into DNA or RNA. Ithas been reported, for example, that the mutation rates in lytic RNAviruses (such as influenza A) are higher than in DNA viruses, at about300-fold times higher, Drake, PNAS, USA 90: 4171-4175 (1993).Retroviruses, however, apparently normally mutate at an average rateabout an order of magnitude lower than lytic RNA viruses. Id.

[0247] For example, in the case of HIV, the viral RNA or theincorporated HIV DNA is copied by reverse transcriptase and then DNApolymerase using a PCR reaction with complementary primers and all fourdeoxynucleoside triphosphates. The region of the genome copiedcorresponds to a 600 nucleotide segment in the reverse transcriptasegene. The copied DNA or RNA after 70 rounds of PCR is treated withrestriction enzymes that cleave the primer sequences, and ligated into aplasmid. After transfection of E. coli, individual clones are obtainedand the amplified segment within the plasmid is sequenced. Mutationswithin this region are determined by computer-aided analysis, comparingthe individual sequences with control viral sequences obtained byparallel culturing of the same virus in the absence of the RNA analog.For each nucleotide, determinations are carried out after ten sequentialrounds of viral passage or at the point of extinction for viraldetection. Analogous procedures would be effective for other viruses ofinterest and would be readily apparent to those of skill in the art.

[0248] Incorporation of an analog by a cellular or viral RNA polymerase,by reverse transcriptase (or other viral enzyme) or by DNA polymerasemay be compared directly, or separately and the separate test resultssubsequently compared. A comparison of incorporation of analogs amongthe polymerases of interest can be carried out using a modification ofthe “minus” sequencing gel assay for nucleotide incorporation. A 5′-³²P-labeled primer is extended in a reaction containing three of thefour nucleoside triphosphates and an analog in the triphosphate form.The template can be either RNA or DNA, as appropriate. Elongation of theprimer past the template nucleotide that is complementary to thenucleotide that is omitted from the reaction will depend and beproportional to the incorporation of the analog. The amount ofincorporation of the analog is calculated as a function of the percentof oligonucleotide that is extended on the sequencing gel from oneposition to the next. Incorporation is determined by autoradiographyfollowed by either densitometry or cutting out each of the bands andcounting radioactivity by liquid scintillation spectroscopy. Those ofskill in the art will recognize that similar experiments can be done todetermine the incorporation of the compounds of the present inventioninto nucleic acids of cancer cells.

[0249] When a nucleoside or nucleotide analog of the invention isadministered to virally infected cells, either in vitro or in vivo, apopulation of cells is produced comprising a highly variable populationof replicated homologous viral nucleic acids. This population of highlyvariable cells results from administering mutagenic nucleoside ornucleotide analogs to virally infected cells and increasing the mutationrate of the virus population. Thus, the highly variable population ofviruses is an indicator that the mutation rate of the virus wasincreased by the administration of the nucleoside or nucleotide analogs.Measuring the variability of the population provides an assessment ofthe viability of the viral population. In turn, the viability of theviral population is a prognostic indicator for the health of the cellpopulation. For example, low viability for an HIV population in a humanpatient corresponds to an improved outlook for the patient.

[0250] In some embodiments, the mutagenic nucleoside or nucleotideanalog of choice will be water-soluble and have the ability to rapidlyenter the target cells. Lipid soluble analogs are also encompassed bythe present invention. The nucleoside or nucleotide analog will bephosphorylated by cellular kinases, if necessary, and incorporated intoRNA or DNA.

[0251] Assays of Viral Replication

[0252] Those of skill in the art recognize that viral replication orinfectivity correlates with the ability of a virus to cause disease.That is, a highly infectious virus is more likely to cause disease thana less infectious virus. In a preferred embodiment, a virus that hasincorporated mutations into its genome as a result of treatment with thecompounds of this invention will have diminished viral infectivitycompared to untreated virus. Those of skill in the art are aware ofmethods to assay the infectivity of a virus. (See, e.g., Condit,Principles of Virology, in Fields Virology, 4th Ed. 19-51 (Knipe et al.,eds., 2001)).

[0253] For example, a plaque-forming assay can be used to measure theinfectivity of a virus. Briefly, a sample of virus is diluted intoappropriate medium and serial dilutions are plated onto confluentmonolayers of cells. The infected cells are overlaid with a semisolidmedium so that each plaque develops from a single viral infection. Afterincubation, the plates are stained with an appropriate dye so thatplaques can be visualized and counted.

[0254] Some viruses do not kill cells, but rather transform them. Thetransformation phenotype can be detected, for example formation of fociafter loss of contact inhibition. The virus is serially diluted andplated onto monolayers of contact inhibited cells. Foci can be detectedwith appropriate dye and counted to determine the infectivity of thevirus.

[0255] Another method to determine infectivity of viruses is theendpoint method. The method is appropriate for viruses that do not formplaques or foci, but that do have a detectable pathology or cytopathiceffect (CPE) in cultured cells, embryonated eggs, or animals. A numberof phenotypes are measurable as CPE, including rounding, shrinkage,increased refractility, fusion, syncytia formation, aggregation, loss ofadherence or lysis. Serial dilutions of virus are applied to anappropriate assay system and after incubation, CPE is assayed.Statistical methods are available to determine the precise dilution ofvirus required for infection of 50% of the cells. (See, e.g., Spearman,Br. J. Psychol. 2:227-242 (1908); and Reed and Muench, Am. J. Hyg.27:493-497 (1938)).

[0256] The ability of a drug to inhibit viral replication or infectivityis expressed as the EC₅₀ of the drug, or the effective concentrationthat prevents 50% of viral replication. Methods described above todetermine the infectivity of a virus are useful to determine the EC₅₀ ofa drug.

[0257] The ability of a drug to kill cells is expressed as the IC₅₀, orthe concentration of drug that inhibit cellular proliferation. Methodsto determine the IC₅₀, of a drug are known to those of skill in the artand include determination of cell viability after incubation with arange of concentrations of the drug.

[0258] Treatment of HIV Strains Resistant to Nucleoside ReverseTranscriptase Inhibitors

[0259] The compounds of the invention can be used to treat HIVinfections and other retroviral infections. The compounds of the presentinvention are particularly well suited to treat HIV strains that areresistant to nucleoside reverse transcriptase inhibitors.

[0260] As of 2001, sixteen antiviral drugs were approved for thetreatment of HIV infection. Seven are nucleoside/nucleotide analog chainterminators or nucleoside reverse trascriptase inhibitors (NRTI), sixare protease inhibitors, and three are non-nucleoside reversetranscriptase inhibitors (NNRTI).

[0261] Until recently, zidovudine was the mainstay of anti HIV drugs.The administration of zidovudine to patients with advanced HIV diseasehas been shown to prolong survival, to improve neurologic function, totransiently improve CD4+ lymphocyte counts, and to decrease the rate ofantigenemia. However, the short-term benefits observed with zidovudinemonotherapy, together with the emergence of zidovudine resistance duringchronic treatment suggested that combination chemotherapy would berequired for prolonged control of HIV infection (see e.g., Loveday etal., Lancet. 345: 820-824 (1995); Volberding, et al., J. Infect. Dis.171: S150-S154. (1995)).

[0262] In 1996, clinical trial results demonstrated that proteaseinhibitors could dramatically reduce the amount of HIV in a patient'sblood and in combination therapy regimens could, in some cases, resultin undetectable viral RNA by PCR. A combination chemotherapy clinicaltrial of saquinavir, zidovudine and zalcitabine demonstrated increasedCD4+ counts and decreased viral burden that were significantly greaterthan a two drug regimen (see, e.g., Collier et al., New Engl. J. Med.334: 1011-1017 (1996)). However, as with nucleoside analogs, there isevidence that cross-resistance develops to protease inhibitors (seee.g., Condra et al., Nature 374: 569-571 (1995)). In fact, simultaneousmutations of the HIV genome coding for resistance to protease inhibitorsand NRTI have been described (Shafer et al., Ann. Intern. Med. 128:906-911 (1998)). Of note, combination therapy regimens (highly activeantiretroviral therapy or HAART), typically initiated with triple drugtherapy, are expensive and because of their complexity and side effectsadversely affect the patients' quality of life. Full therapeutic benefitmay require near perfect adherence to the dosage, frequency, timing anddietary restrictions of many agents (see, e.g., Stone Clin. Infect. Dis.33: 865-872 (2001)). Furthermore, if virologic, immunologic or clinicalfailure develops during triple therapy a regimen of five or more drugsmay be necessary, so called mega-HAART (BHIVA Writing Committee. HIVMed. 1: 76-101 (2000)).

[0263] Thus, novel HIV therapeutics with a low likelihood of viralresistance are required in the marketplace. One embodiment of thisinvention describes a novel class of nucleoside and nucleotide analogsfor activity against a panel of HIV strains resistant to conventionalNRTI.

[0264] Routine screening of candidate 5-aza-dC formulations andderivatives was performed against HIV LAI. Candidates with high activityagainst HIV LAI were also screened for activity against strains of HIVwith preexisting resistance to nucleoside reverse transcriptaseinhibitors (NRTI).

[0265] HIV strains resistant to NRTI are known and mutations in thereverse transcriptase (RT) enzyme responsible for the resistance havebeen analyzed. Resistance mutations in HIV RT appear to only increasethe pre-existing capabilities of wild type RT rather than creating newones. Two mechanisms of resistance toward NRTI have been described: anincrease in efficiency of discrimination between an NRTI and a naturallyoccurring nucleoside, and excision of an NRTI by pyrophosphorolysis inthe presence of nucleotides (see, e.g., Isel et al., J. Biol. Chem. 276:48725-48732 (2001)). Decrease in affinity of HIV RT for a NRTI usuallyinvolves alterations in the sugar moiety of an analog, e.g., mutationsM184V or Q151 M (see, e.g., Sluis-Cremer et al., Cell. Mol. Life Sci.57: 1408-1422 (2000)). Alternatively, chain terminators may be removedby pyrophosphorolysis, or reverse nucleotide polymerization, wherepyrophosphate acts as acceptor molecule for the removal of the chainterminator. Removal of the chain-terminator frees RT to incorporate thenatural nucleotide substrate and rescue viral replication. ATP has alsobeen proposed as an acceptor molecule for the removal ofchain-terminators and is referred to as primer unblocking (see, e.g.,Naeger et al., Nucleosides Nucleotides Nucleic Acids 20: 635-639(2001)).

[0266] Viral resistance is less likely to emerge after treatment withmutagenic nucleotide analogues than after treatment with NRTI. Forexample, mutagenic nucleotide analogues apply less selective pressure toa viral population for emergence of resistant variants than approvedantivirals, which attempt to immediately halt viral replication.Mutagenic nucleotide analogues adversely affect all viral proteins.Decreased affinity of HIV RT for a modified nucleoside sugar is onemechanism of viral resistance. Mutagenic nucleotide analogues haveunmodified sugars. For example, it has been shown that RT may recognizethe absence of a 3′-OH group, resulting in cross-resistance among chainterminators (see, e.g., Huang et al., Science 282: 1669-75 (1998)).Mutagenic nucleotide analogues, like natural ds, have a 3′-OH. Becausemutagenic nucleotide analogues do not terminate replication,pyrophosphorolysis, the other principal mechanism of viral resistance toconventional nucleoside analogs, is unlikely to be applicable to MDRN.Pyrophosphorolysis by RT results in the excision of a chain terminatorpreventing DNA chain elongation.

[0267] Cross resistance between NRTI and mutagenic nucleoside ornucleotide analogues can be tested by determining the EC₅₀ for amutagenic nucleoside or nucleotide analogue in a wild-type HIV strainand in an HIV strain resistant to one or more NRTI's. If the EC₅₀ forthe mutagenic nucleoside or nucleotide analogue is higher in the NRTIresistant strain than in the wild-type strain, it suggests thatcross-resistance has occurred. Experiments have demonstrated thatcross-resistance is unlikely to develop between NRTI and mutagenicnucleoside or nucleotide analogues. A panel of three HIV NRTI resistantstrains (AIDS Research and Reference Reagent Program, Division of AIDS,NIAID, NIH), where resistance is achieved by pyrophosphorolysis orenhanced RT discrimination, were used to test the effectiveness of5-aza-2′-deoxycytidine (5-aza-dC), a mutagenic nucleoside or nucleotideanalogue. These strains have most of the mutations in susceptibility toNRTI present in routine clinical samples (see, e.g., Hertogs,. AntiviralDrug Discovery and Development Summit. Strategic Research Institute, NY,N.Y. (2001)), namely: 1) HIV-1 LA1-M184V: The M184V mutation confersresistance to lamivudine (3TC). M184V also decreases the likelihood ofincorporation of 3TC-TP by interaction with the sulfur of theoxathiolane ring but interestingly also enhances sensitivity tozidovudine perhaps by reducing pyrophosphorolytic activity (see e.g.,Boyer et al., J. Virol. 76: 3248-3256 (2002)). 2) HIV-1 RTMDR1, with74V, 41L, 106A and 215Y mutations. RTMDR1 is resistant to zidovudine,didanosine, nevirapine and other non-nucleoside reverse transcriptaseinhibitors. Template/primer repositioning may play a role in thedecreased DNA synthesis processivity associated with the 74V mutationfor didanosine. Resistance mutations 41L and 215Y enhancepyrophosphorolysis (see, e.g., Sluis-Cremer et al., supra). 3) HIV-1RTMC, with 67N, 70R, 215F and 219Q mutations. RTMC is resistant tozidovudine. All of these mutations enhance pyrophosphorolysis (Id.). TheEC₅₀ of 5-aza-dC for the wild-type HIV strain LAI was similar to theEC₅₀ of 5-aza-dC for NRTI resistant strains. In contrast, the EC₅₀ ofAZT or 3TC for the wild-type HIV strain LAI was markedly different thanthe EC₅₀ of AZT or 3TC for the appropriate NRTI resistant strain (e.g.,RTMC, M184V, or RTMDR1). Other NRTI mutants are available and can beassayed in a similar manner (Gonzales et al., Program and Abstracts ofthe Forty-Second Interscience Conference on Antimicrobials andChemotherapy. Abstract No. 3300 (2002)). Mutations include: M41L, E44D,A62V, K65R, D67N, T69DN, T69S_SS, K70R, L74V, V751, F77L, Y115F, F116Y,V1181, Q151M, M184V, L210W, T215F and K219QE.

[0268] Treatment of Cancer

[0269] The compounds of the present invention can be used to treatcancer. Because malignant cells replicate more rapidly than nonmalignantcells, the compounds of the invention are preferentially incorporatedinto malignant cells. In a preferred embodiment, leukemias and otherhematopoetic cancers are treated using the compounds of the presentinvention. Without wishing to be bound by theory, the nucleoside andnucleotide analogues of the present invention are incorporated into thenucleic acids of a cancerous cell, either DNA or RNA. The nucleoside andnucleotide analogues have phosphodiester linkages or obtainphosphodiester linkages, allowing them to be incorporated and extendedby a polymerase. In one embodiment, the nucleoside and nucleotideanalogues have altered base-pairing properties allowing incorporation ofmutations into the genome of the cancer cell, dramatically increasingthe mutation rate in the cancer cell. The increased mutation rateresults in decreased viability of progeny cells, leading to death of thecancer cells, or a diminished growth rate, or inability to metastasize.In another embodiment, mutations are incorporated into transcriptionproducts, e.g., mRNA molecules that encode proteins or tRNA moleculesuseful for translation of proteins. The mutated transcription productsencode mutated proteins, for example, proteins with altered amino acidsequences or trancations that lead, in turn to the inactivation of theprotein. The inability of the cancer cell to consistently encode activeprotein can also result in death of the cancer cells, a diminishedgrowth rate, inability to metastasize, or inability to proliferate.

[0270] Those of skill in the art are aware of methods to test theeffectiveness of compounds in treating cancer. For example, cancer cellsof interest can be grown in culture and incubated in the presencevarying concentrations of the compounds of the present invention.Frequently, uptake of vital dyes, such as MTT, is used to determine cellviability and cell proliferation. When inhibition of cell proliferationis seen, the IC₅₀ of the compound can be determined, essentially asdescribed above. Those of skill in the art will also know to test thecompounds of the present invention in animal models, for example, nudemice injected with transformed cells. The data gathered in tissueculture models and animal models can be extrapolated by those of skillin the art for use in human patients.

[0271] Combination Therapies

[0272] The compounds of the invention can also be used in combinationwith other drugs to treat viral diseases or cancers.

[0273] For example, mutagenic nucleoside analogs can be used incombination with other antiviral therapies, such as nucleoside reversetranscriptase inhibitors, (e.g., Zidovudine (ZDV or AZT), Didanosine(ddI), Zalcitabine (ddC), Stavudine (d4T), Lamivudine (3TC), Abacavir(ABC), and Tenofovir tenofovir disoproxil fumarate (TDF)),non-nucleoside reverse transcriptase inhibitors, (e.g., Nevirapine(NVP), Delavirdine (DLV), and Efavirenz (EFV)), protease inhibitors,(e.g., Invirase, Fortovase, Norvir, Crixivan, Viracept, Agenerase,Kaletra, Reyataz, fosamprenavir, and tipranavir) integrase inhibitors,fusion inhibitors or immunomodulators, such as interferon. Drugs thatinduce viral replication, such as diacylglycerol analogues, (e.g., Hameret al. Journal of Virology. 77:10227-10236 (2003)), might also benefitfrom combination with a viral mutagen. These drugs may have utility indecreasing the size of the viral reservoir. Mutagenic nucleosideanalogues can also be used in combination with cytokines such as IL-2.(See, e.g., Kedzierski and Crowe, Antiviral Chem. & Chemo. 12:133-150(2001)). Combination of such compounds with a viral mutagen, would allowincorporation of mutagenic nucleosides into the viral genome producingless fit viruses and ultimately resulting in viral extinction.

[0274] For cancer treatment mutagenic nucleoside analogs can be used incombination with other anticancer therapies, e.g. radiation,chemotherapeutic agents, hormone analogues, immunostimulants,interferons, cytokines, and antibodies.

[0275] Administration

[0276] The pharmaceutical preparation is preferably in unit dosage form.In such form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

[0277] The compounds (in the form of their compositions) areadministered to patients by the usual means known in the art, forexample, orally or by injection, infusion, infiltration, irrigation, andthe like. For administration by injection and/or infiltration orinfusion, the compositions or formulations according to the inventionmay be suspended or dissolved as known in the art in a vehicle suitablefor injection and/or infiltration or infusion. Such vehicles includeisotonic saline, buffered or unbuffered and the like. Depending on theintended use, they also may contain other ingredients, including otheractive ingredients, such as isotonicity agents, sodium chloride, pHmodifiers, colorants, preservatives, antibodies, enzymes, antibiotics,antifungals, antivirals, other anti-infective agents, and/or diagnosticaids such as radio-opaque dyes, radiolabeled agents, and the like, asknown in the art. However, the compositions of this invention maycomprise a simple solution or suspension of a compound or apharmaceutically acceptable salt of a compound, in distilled water orsaline.

[0278] Alternatively, the therapeutic compounds may be delivered byother means such as intranasally, by inhalation, or in the form ofliposomes, nanocapsules, vesicles, and the like. Compositions forintranasal administration usually take the form of drops, sprayscontaining liquid forms (solutions, suspensions, emulsions, liposomes,etc.) of the active compounds. Administration by inhalation generallyinvolves formation of vapors, mists, dry powders or aerosols, and againmay include solutions, suspensions, emulsions and the like containingthe active therapeutic agents

[0279] Routes and frequency of administration of the therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, and may be readily established using standardtechniques. Preferably, between 1 and 100 doses may be administered overa 52-week period. When treating a viral disease, a suitable dose is anamount of a compound that, when administered as described above, iscapable of killing or limiting the infectivity of a virus. When treatingcancer, a suitable dose is an amount of a compound that, whenadministered as described above, is capable of killing or slowing thegrowth of, cancers or cancer cells.

[0280] In general, an appropriate dosage and treatment regimen providesthe active compound(s) in an amount sufficient to provide therapeuticand/or prophylactic benefit. A response can be monitored by establishingan improved clinical outcome (e.g., longer viral disease-free survivalor in cancer patients, more frequent remissions, complete or partial, orlonger disease-free survival) in treated patients as compared tonon-treated patients.

[0281] A therapeutic amount of a compound described in this application,means an amount effective to yield the desired therapeutic response, forexample, an amount effective to kill or limit the infectivity of avirus, when treating a viral disease. When treating a patient withcancer, a therapeutic amount of a compound described in this applicationis for example, an amount effective to delay or halt the growth of acancer or to cause a cancer to shrink or not metastasize. For treatmentof both viral diseases and cancer, if what is administered is not thecompound (or compounds), but an enantiomer, prodrug, salt or metaboliteof the compound (or compounds), then the term “therapeutically effectiveamount” means an amount of such material that produces in the patientthe same blood concentration of the compound in question that isproduced by the administration of a therapeutically effective amount ofthe compound itself. Similarly, if an enantiomer, prodrug or metaboliteof the compositions, or a salt of the compositions or of any of theseother compounds, is being administered, then one therapeuticallyeffective amount of such a compound is that amount that produces atherapeutically relevant blood concentration of the compositions in apatient. Oral dosages optimally range from 500 mg to 2 grams fortreatment of viral diseases or cancer. Those of skill in the art areaware of the routine experimentation that will produce an appropriatedosage range for a patient in need of treatment by oral administrationor any other method of administration of a drug, e.g., intravenousadministration or parenteral administration, for example. Those of skillare also aware that results provided by in vitro or in vivo experimentalmodels can be used to extrapolate approximate dosages for a patient inneed of treatment.

[0282] Patients that can be treated with the a compound described inthis application, and the pharmaceutically acceptable salts, prodrugs,enantiomers and metabolites of such compounds, according to the methodsof this invention include, for example, patients that have beendiagnosed as having HIV infection, hepatitis B, hepatitis C, or smallpox or vaccinia virus.

[0283] Other patients that can be treated with the a compound describedin this application, and the pharmaceutically acceptable salts,prodrugs, enantiomers and metabolites of such compounds, according tothe methods of this invention include, for example, patients that havebeen diagnosed as having lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head and neck, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer or cancer of theanal region, stomach cancer, colon cancer, breast cancer, gynecologictumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina or carcinoma of the vulva), Hodgkin's disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrine system(e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomasof soft tissues, cancer of the urethra, cancer of the penis, prostatecancer, chronic or acute leukemia, solid tumors of childhood,lymphocytic lymphomas, cancer of the bladder, cancer of the kidney orureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), orneoplasms of the central nervous system (e.g., primary CNS lymphoma,spinal axis tumors, brain stem gliomas or pituitary adenomas).

[0284] In further aspects of the present invention, the compositionsdescribed herein may be used to treat hematological malignanciesincluding adult and pediatric AML, CML, ALL, CLL, myelodysplasticsyndromes (MDS), myeloproliferative syndromes (MPS), secondary leukemia,multiple myeloma, Hodgkin's lymphoma and Non-Hodgkin's lymphomas.

[0285] Within such methods, pharmaceutical compositions are typicallyadministered to a patient. As used herein, a “patient” refers to anywarm-blooded animal, preferably a human.

[0286] Kits for administering the compounds may be prepared containing acomposition or formulation of the compound in question, or anenantiomer, prodrug, metabolite, or pharmaceutically acceptable salt ofany of these, together with the customary items for administering thetherapeutic ingredient.

[0287] All references and patent publications referred to herein arehereby incorporated by reference herein. As can be appreciated from thedisclosure provided above, the present invention has a wide variety ofapplications. Accordingly, the following examples are offered forillustration purposes and are not intended to be construed as alimitation on the invention in any way.

EXAMPLES Example 1 5-aza-dC is a Potent Mutagen of HIV

[0288] 5-aza-2′-deoxycytidine (5-aza-dC) is a potent viral mutagen thatis capable of eradicating HIV in a single passage.

[0289] Viral Stocks for Test of Antiviral Activity

[0290] The strains of HIV-1 used for primary drug screening are HIV-1LAI or the appropriate strain of NRTI resistant HIV for studies ofcross-resistance. Virus was propagated on MT-2 cells at an multiplicityof infection (MOI) of 0.01 to generate virus stocks. Briefly, the MT-2cells were suspended in RPMI 1640 media supplemented with 10% fetalbovine serum, streptomycin and penicillin (cRPMI) and grown in a 37° C.incubator containing 5% CO₂. Serial dilution of the virus and infectionof MT-2 cells were followed by an ELISA detecting the capsid protein ofHIV-1 (p24) and used to determine the titer of the virus stocks (50%tissue culture infectious dose (TCID₅₀)). The ELISA was performedaccording to the manufacturer's instructions. The MT-2 cells are alsoused for visualizing the cytopathic effects of HIV-1 growth (e.g.syncytia formation).

[0291] Treatment of HIV-1-Infected Cells with Mutagenic Nucleoside orNucleotide Analogues

[0292] 0.1 ml of a 3×10⁵ cells/ml MT-2 cell suspension were seeded in96-well plates at 3×10⁴ cells/well. The compounds to be assayed werediluted in a separate 96-well at ten times (10×) the concentrationneeded for the screen. 22 μl of the 10× compounds was then added to thewells in triplicate except for six control wells, containing uninfectedMT-2 cells alone (3 wells) and untreated HIV-infected MT-2 cells (3wells). This was followed by the addition of HIV-1 at an MOI of 0.01,except for the three wells serving as the uninfected control. 0.1 ml ofcRPMI was added to the uninfected well instead of virus. After theaddition of virus, the 96-well plate was centrifuged at 1,200×g for twohours to enhance the adsorption of the virus by the MT-2 cells (see,e.g., O'Doherty, ^(J. Virol.) 74:10074-10080 (2000)). After thecentrifugation step, the 96-well plate was then incubated in a 37° C.incubator containing 5% CO2 for three days. At the end of this timeperiod the virus and cells were mixed by gentle pipetting followed by a1 minute spin at 600×g to pellet the cells. The supernatant of each wellwas then serially diluted 1,000-fold into new 96-well plates to serve asinoculum for the next passage and assayed by ELISA for the amount of p24produced. The next passage was performed as described above, except thatthe virus used to infect the cells was derived from the 1,000-folddilution plate. To generate an EC₅₀ value, half-log concentrations ofmutagenic nucleoside or nucleotide analogue capable of eradicating virusin a single passage are tested as above to generate a dose-responsecurve.

[0293] EC₅₀ values were determined for the following compounds:5-aza-dC, 5-aza-dU, DH-aza-dC, and5-methyl-5,6-dihydro-5-azadeoxycytidine (MeDHAdC). 5-aza-dC has an EC₅₀(effective concentration that prevents 50% of viral replication) of 3 nMagainst the wild-type HIV strain LAI. Results are shown in FIG. 4. TheEC₅₀ values for the other compounds are 3 μM for DHAdC and 10 μM forMeDHAdC.

[0294] Assessment of the Frequency of Mutations to the Viral GenomeInduced by Mutagenic Nucleoside Analogues

[0295] The viral genomic DNA from cells treated with thedeoxyribonucleoside analog 5-aza-dC (30 μM) was purified using a QiagenDNeasy® Kit. 1 μg of genomic DNA was used to amplify a 1 kb region ofthe HIV-1 RT proviral DNA by PCR. The PCR product was then cloned into aTOPO® cloning vector. A Millipore Miniprep Kit was used to purifyplasmid containing proviral inserts. About 45 positive clones weresequenced in both directions by a Beckman Coulter CEQ 8000. Thesequencing results were analyzed and assembled using the DNASTAR Stagmanprogram. Each mutated base was counted and the mutation rate wascalculated over the total number of sequenced nucleotides. The resultswere compared with the background mutation rate generated from untreatedcontrol virus and are shown in Table 3. Sixty-one mutations were foundin 26,187 bases sequenced in the 5-aza-dC treated cells. Only onemutation in the drug-free control could be confirmed by sequencing inboth directions and thus, the mutation rate in the control may beoverestimated. Thus, sequencing of a fragment of the nucleic acidencoding HIV reverse transcriptase has confirmed that 5-aza-dC ismutagenic to the viral genome. TABLE 3 MUTA- TIONS/ NUCLEO- GΠA TΠC AΠTAΠC GΠT CΠG ANALOG TIDE % AΠG CΠT TΠA CΠA TΠG GΠC 5-aza- 61/ 0.223 24 68 0 10 13 dC 26,187 None 6/ 0.054 5 1 11,023

[0296] Assessment of Mutagenic Nucleoside Analogue Cytotoxicity

[0297] For each compound, cytotoxicity was evaluated on MT-2 cells. MT-2cells were seeded at 3×10⁴ cells/well in 96-well plates. The cells weretreated with compounds at half-log serial dilutions from 100 μM to 0.32μM in triplicate. After 5 days growth in a 37° C. incubator containing5% CO₂, MTT was added to a final concentration of 0.5 mg/ml and thenincubated for four hours at 37° C. 10% SDS in 0.02 N HCl was added tolyse the cells overnight at 37° C. The plates were read on a TecanGenius microplate reader at wavelengths of 570 nm/650 nm. The doseresponse curve was graphed by comparing the treated cells with theuntreated control and the IC₅₀ was determined for each compound. For5-aza-dC, the IC₅₀ was greater than 10 μM. For DHadC, the IC₅₀ wasgreater than 1 mM. The IC₅₀ for 5-Me-DHAdC was not determined.

Example 2 5-aza-dC is Effective Against Wild-Type HIV Strains and NRTIResistant HIV Strains

[0298] Assessment of Sensitivity of NRTI-Resistant HIV Strains toMutagenic Nucleoside Analogue

[0299] To determine if there is resistance of HIV NRTI resistant strainsto mutagenic nucleoside analogues, NRTI resistant strains were grown inthe presence of 5-aza-dC to determine whether the EC₅₀ for 5-aza-dC isdifferent from the WT strain (HIV-1 LAI). An EC₅₀ higher for theNRTI-resistant strains than for the WT strain suggests that there iscross-resistance between 5-aza-dC and the particular NRTI. The EC₅₀experiment was performed in a similar manner described above for thedrug screen against HIV-LAI. Growth of HIV NRTI resistant strains in thepresence of the appropriate concentration of NRTI was used as a positivecontrol.

[0300] Three HIV NRTI resistant strains (HIV-1 LAI-M184V, HIV-1 RTMDR1,with 74V, 41L, 106A and 215Y mutations, and HIV-1 RTMC, with 67N, 70R,215F and 219Q mutations) were used to test the effectiveness of5-aza-dC. Results are shown in Table 4. These experiments demonstratethat HIV strains with resistance to NRTI are not cross-resistant with5-aza-dC. The EC₅₀ of 5-aza-dC for the wild-type HIV strain LAI wassimilar to the EC₅₀ of 5-aza-dC for NRTI resistant strains. In contrast,the EC₅₀ of AZT or 3TC for the wild-type HIV strain LAI was markedlydifferent than the EC₅₀ of AZT or 3TC for the appropriate NRTI resistantstrain (e.g., RTMC, M184V, or RTMDR1). Other NRTI mutants are availableand can be assayed in a similar manner (Gonzales et al., Program andAbstracts of the Forty-Second Interscience Conference on Antimicrobialsand Chemotherapy. Abstract No. 3300 (2002)). Mutations include: M41L,E44D, A62V, K65R, D67N, T69DN, T69S_SS, K70R, L74V, V751, F77L, Y115F,F116Y, V1181, Q151M, M184V, L210W, T215F and K219QE. TABLE 4 5-aza-dCAZT 3TC HIV Strain (EC₅₀) nM (EC₅₀) nM (EC₅₀) nM LAI (wild type) 3 10 45RTMC 5 300 330 M184V 10 10 >32,000 RTMDR1 10 60 N.D.

[0301] Table 4: EC₅₀'s of 5-aza-dC versus zidovudine (AZT) andlamivudine (3TC) against wild type HIV LAI and drug resistant strains.

Example 3 5-aza-C is Effective Against Riboviruses

[0302] 5-aza-C was effective against two model riboviruses: measlesvirus and bovine viral diarrhea virus.

[0303] Viral Stocks for Test of Antiviral Activity

[0304] Measles virus (MV) and bovine viral diarrhea virus (BVDV) aremembers of two distinct ribovirus families, Paramyxoviridae andFlaviviridae. For primary screening, drug activities were tested againstthese two viruses.

[0305] MV Nagahata strain was used for drug testing. Compared to somelaboratory strains, this virus strain replicates lytically in primaryhuman embryonic lung cells and causes extensive cytopathic effect duringacute infection. The virus stock was prepared by growing the virus onCV-1 cells at a MOI of 0.01. The titer of the virus stock was determinedby plaque formation assay after series dilution.

[0306] The BVDV strain used for drug testing was the Singer strain. Thisvirus also causes a cytopathic effect that is measurable and allowsestimation of the level of infection. The visible cytopathology can beused as an endpoint for titrating the virus by 50% tissue cultureinfectious dose (TCID₅₀). The BVDV was propagated in bovine turbinate(BT cells). The virus TCID₅₀ was determined by counting the cytopathiceffect at the endpoint dilution. Briefly, confluent BT cells in 96-wellplates were infected with the virus at 8 independent serial 10-folddilutions. All the plates were incubated for five days at 37° C. in 5%CO₂. Each well was scored as positive or negative appearance of visiblecytopathic effect. The titers were calculated by the method of Reed andMuench, Am. J. Hyg. 27:493-497 (1938), and the mean titer and standarddeviation for each of the 3 replicates for each drug concentration andthe positive and negative control were calculated.

[0307] Treatment of MV- or BVDV-Infected Cells with Mutagenic NucleosideAnalogs

[0308] Virus susceptible cells (CV-1 or BT cells) were seeded in 96-wellplates at 2×10⁴ cells/well. The virus was inoculated onto the cellmonolayers at a MOI of 0.001˜0.002 to keep 30 plaque forming units (pfu)in each well. The inoculum was maintained in 37° C. for about one hourand the supernatant was discarded. Fresh media containing appropriatedrug concentration was added in each well. Untreated control was run inparallel. Each drug was tested in triplicate. Three days afterinfection, cytopathic effect (CPE) was examined in the untreated controlwells. When the untreated control cells showed more than 90% CPE, theinfected cells were harvested. The plates went through one “freeze-thaw”cycle to release intracellular virus. The virus stock was saved for nextround of passage. The virus titer was determined as described above.Virus at a titer of 10⁵˜10⁶ pfu/ml was produced by this method.

[0309] The following results were obtained. 5-aza-C was effectiveagainst BVDV as a surrogate for hepatitis C virus, with an EC50 of 10μM. 5-aza-C was effective against measles virus with an EC₅₀ of 15 μM.

[0310] Assessment of the Frequency of Mutations to the Viral GenomeInduced by Ribonucleoside Analogs.

[0311] The mutagenicity of test analogs showing antiviral activitieswill be evaluated. Studies with ribavirin indicate that the mutationrate is related to the concentration of introduced analogs (see e.g.,Crotty et al., Nature Med. 8(12):1375-1379 (2000)). In order to shortenthe duration of the experiment, the infected cells are treated with testanalogs at 2 mM so that a high mutation rate can be achieved. Thisdosage is usually toxic to cells and no progeny virus is produced. Cellsare inoculated with BVDV or MV and incubated for 4 hours. The incubationallows the virus to express the proteins necessary for viral RNAreplication. The test analogs are then added and incubated for another24 hours. Viral RNA is extracted with a Qiagen RNeasy Kit. About 1 μg ofRNA is primed with viral gene specific oligonucleotides and cDNA will besynthesized using M-MLV reverse transcriptase. About 1 kb fragmentscovering BVDV variable region E2, and conserved region NS3 is amplifiedfrom 1 μg cDNA by PCR. The PCR products is cloned into an InvitrogenTopo© cloning vector. A Millipore Miniprep kit is used to screenplasmids containing viral inserts. About 40 positive clones aresubjected to sequence reaction. Each sample is sequenced from twodirections. Sequence results were assembled by using the DNASTAR Stagmanprogram. Each mutated base is counted and mutation rate is calculatedover the total number of sequenced nucleotides. The results are comparedwith the background control generated from untreated control virus.

[0312] Assessment of Cytotoxicity

[0313] For each test analog, cytotoxicity is evaluated on BT cells. BTcells are seeded at 1×10⁴, 4×10⁴, 1×10⁵ cells/well in 24-well plates.The next day the cells are treated with drugs at concentrations of 0,100 μM, 300 μM and 1,000 μM. Each dose is tested in duplicate. After thecells are grown for three generations, MTT at final concentration of 1μg/μl is added in the media and the cells are incubated for three hours.10% SDS in 0.02N HCl will be added to lyse the cells overnight. Theplates are read on a Tecan Genius microplate reader at wavelengths of570/650 nm. The dose response curve is graphed by comparing the treatedcells with the untreated control. The dosage that inhibits 50% of cellgrowth (IC₅₀) is then determined. For those analogs showing antiviralactivity, cytotoxicity is further examined in T lymphoid CEM cells byusing the same method.

Example 4 Synthesis

[0314] Materials and Methods

[0315] 5-Azacytidine (1) and 2′-deoxy-5-azacytidine (2) (Scheme 1) arecommercially available from Sigma.

[0316] 5-Azauridine (3)

[0317] Compound 3 was synthesized following a literature procedure(Nucl. Acid Chemistry (L. B. Townsend and R. S. Tipson Eds) Part 1, p455-459, N-Y 1978).

[0318] 5,6-Dihydro-5-azauridine (4)

[0319] Compound 4 was synthesized from 5-azauridine according to(Piskala A., {haeck over (C)}esneková B., Veselý J. Nucl. Acids Symp.Ser. No 18 (1978) pp 57-60).

Example 4a Synthesis of1-(2-Deoxy-3,5-di-O-p-toluoyl-β-D-ribofuranosyl)-4-amino-1,2-dihydro-1,3,5-triazin-2-one(5)

[0320] The desired compound was synthesized by (Niedballa U., VorbrüggenH. J. Org. Chem. Vol. 39, No. 25, 1974, pp 3672-3674) as a whitecrystalline powder, m.p. 195-196° (from ethyl acetate).

Example 4b Synthesis of1-(2-Deoxy-3,5-di-O-p-toluoyl-α-D-ribofuranosyl)-4-amino-1,2-dihydro-1,3,5-triazin-2-one(6)

[0321] The desired compound was isolated as a side reaction product fromthe above synthesis of compound 5 as a white crystalline powder, m.p.210-211° (from CH₂Cl₂-hexanes), 220° (from ethanol).

[0322] NMR (DMSO-d₆) δ 8.44 (s, 1H, H-6), 7.91 (d, 2H, Ar), 7.71 (d, 2H,Ar), 7.51 (d J=8.7 Hz, 2H, NH₂), 7.36 (d, 2H, Ar), 7.28 (d, 2H, Ar),6.11 (d J=5.7 Hz, 1H, H-1′), 5.53 (d, 1H, H-4′), 5.11 (t, 1H), 4.45 (d,2H), 2.86 (m, 1H), 2.50 (m, 1H), 2.39 (s, 3H, Me), 2.37 (s, 3H, Me). NMR(CDCl₃) δ 8.25 (s, 1H, H-6), 7.92 (d, 2H, Ar), 7.69 (d, 2H, Ar), 6.88(br s, 2H, NH₂), 7.26 (d, 2H, Ar), 7.19 (d, 2H, Ar), 6.27 (d J=6.3 Hz,1H, H-1′), 5.61 (d, 1H, H-4′), 4.88 (t, 1H), 4.55 (d, 2H), 3.00-2.87 (m,1H), 2.67 (m, 1H), 2.42 (s, 3H, Me), 2.38 (s, 3H, Me).

Example 4c Synthesis of1-(2-Deoxy-3,5-di-O-p-toluoyl-β-D-ribofuranosyl)-4-amino-1,2,5,6-tetrahydro-1,3,5-triazin-2-one(7) (Scheme 2)

[0323] To a suspension of 5 (0.50 g, 1.08 mmol) in acetic acid (5 mL)was added NaBH₄ in 2 portions (2×0.040 g, 2.11 mmol) with ice cooling in15 min interval. The mixture was stirred for another 15 min at 0° C. andevaporated. The residual oil was suspended in CHCl₃ (150 mL), washedwith sat. NaHCO₃ solution (70 mL) and dried over Na₂SO₄. The solutionwas filtered through Celite, concentrated by partial evaporation andapplied to a silica gel column (1.5×22 cm). The column was eluted withCH₂Cl₂— MeOH mixtures (5-15% v/v MeOH, 700 mL). The product fractionswere combined and evaporated, crystallized from MeOH-ether (1:2) giving0.3 g of 7 as a solid. MS ES⁺467 [M+H⁺].

Example 4d Synthesis of1-(2-Deoxy-3,5-di-O-p-toluoyl-α-D-ribofuranosyl)-4-amino-1,2,5,6-tetrahydro-1,3,5-triazin-2-one(8)

[0324] Compound 8 was synthesized by analogy to 7 starting from 6. NMR(DMSO-d₆) δ 7.92-7.85 (m, 4H, Ar), 7.38-7.30 (m, 4H, Ar), 6.27 (ddJ=8.2+4.8 Hz, 1H, H-1′), 5.46 (m, 1H, H-4′), 4.58 (q, 2H, CH₂), 4.56 (m,1H), 4.37 (m, 2H), 3.5 (br s, 3H, NH, NH₂), 2.78 (m, 1H), 2.50 (m, 1H),2.39 (s, 3H, Me), 2.38 (s, 3H, Me). MS ES⁺467 [M+H⁺].

Example 4e Synthesis of1-(2-Deoxy-β-D-ribofuranosyl)-4-amino-1,2,5,6-tetrahydro-1,3,5-triazin-2-one(2′-deoxy-5,6-dihydro-5-azacytidine) (9)

[0325] Method A (Reduction of 2). To a suspension of2′-deoxy-5-azacytidine (2) (0.045 g, 0.2 mmol) in 96% ethanol (2.9 mL)was added NaBH₄ (40 mg, 1.06 mmol), and the mixture was stirred for 10min at RT. Water (4 mL) was added to the mixture giving a clear solutionthat was directly used for RP HPLC purification using a gradient of MeCNin 0.1 M triethylammonium bicarbonate buffer. The main fraction afterevaporation provided 0.04 g of 9 as a solid. MS ES⁺231 [M+H⁺].

[0326] Method B (Deprotection of 7, scheme 3). To a solution of 7 (50mg) in MeOH (5 mL) was added 25% aq. NH₃ (2 mL) giving a suspension thatbecame a solution upon stirring overnight. The mixture was evaporated,redissolved in water and purified by RP preparative HPLC using agradient of MeCN in 0.1 M triethylammonium bicarbonate buffer. The mainfraction after evaporation gave 0.03 g of 9 as a solid. MS ES⁺231[M+H⁺].

[0327] Method C (via reduction of ribo-compound 1, scheme 4, fivesteps).

[0328] Step 1. Synthesis of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5-azacytidine (11)

[0329] Compound 11 was prepared by analogy to the process described forTIPS-protection of compound 2 in (Goggard A. J., Marquez V. E.Tetrahedron Letters, vol. 29, No. 15, 1988, pp 1767-1770). Compound 11was obtained as a colorless solid with m.p. 249°.

[0330] Step 2. Synthesis of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5,6-dihydro-5-azacytidine(12)

[0331] 0.57 g of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5-azacytidine wassuspended in 10 mL of THF under nitrogen. NaBH₄ (0.34 g, 9 mmol, 7.7eq.) was added and the reaction mixture was sonicated for 3 min. Afterstirring for 2 h at room temperature, 100 mL of saturated NaCl was addedand the mixture was extracted 3 times with 150 mL of EtOAc. The organicfractions were dried over Na₂SO₄, filtered and evaporated. The productwas isolated by silica gel LC in EtOAc with MeOH gradient. MS ES⁻ 487.0[M−H⁺], yield of 12 is 0.27 g (47%).

[0332] Step 3. Synthesis of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5,6-dihydro-N⁴-isobutyryl-5-azacytidine(13) (Scheme 5)

[0333] 265 mg of3′,5′-O-(,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5,6-dihydro-5-azacytidinewas dissolved in a 1:1 mix of pyridine and dichloromethane (10 mL) andcooled to 0° C. Chlorotrimethylsilane (344 μL, 5 eq.) was added followedafter 15 min by isobutyryl chloride (341 μL, 6 eq.). After 1.5 h ofstirring the reaction was quenched with 10 mL of MeOH, evaporated,dissolved in EtOAc (100 mL) and extracted twice with saturated NaCl (50mL). The organic layer was dried with Na₂SO₄, evaporated and the residuewas redissolved in MeOH and left overnight at room temperature. Then thesolution was evaporated and the product was isolated by flashchromatography (MeOH gradient in dichloromethane). MS ES⁻ 557.1 [M−H⁺],yield 180 mg (59%).

[0334] Step 4. Synthesis of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-5,6-dihydro-N⁴-isobutyryl-5-azacytidine(14)

[0335] 80 mg of3′,5′-O-(,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5,6-dihydro-N⁴-isobutyryl-5-azacytidinewas dissolved in 4 mL of dry DMF and 1,1′-thiocarbonyldiimidazole (77mg, 3 eq.) was added. After overnight incubation at ambient temperaturethe reaction mixture was diluted with 50 mL of EtOAc and extracted withwater (4×50 mL). The organic fraction was dried over Na₂SO₄, filtered,evaporated to oil, coevaporated with toluene twice and dissolved in 10mL of toluene. The solution was degassed with argon for 45 min, 107 μLof tributyltin hydride (5 eq.) and 13 mg of2,2′-azobis(isobutyronitrile) were added. The reaction mixture washeated at 80° C. for 3 h, cooled, evaporated and separated by flashchromatography on silica gel (MeOH gradient in dichloromethane). Themain product showed expected ES⁺ MS signals at 543.3 [M+H⁺] and 565.5[M+Na⁺], yield 18 mg (23%).

[0336] Step 5. Synthesis of 2′-deoxy-5,6-dihydro-5-azacytidine (9)

[0337] 6 mg of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-5,6-dihydro-N⁴-isobutyryl-5-azacytidinewas dissolved in 2 mL of MeOH and treated with 6 mL of 25% aq. NH₃ over16 h at room temperature. The solution was evaporated to dryness,coevaporated with toluene and dissolved in 2 mL of THF. To the solution0.5 mL of 1 M tetrabutylammonium fluoride was added and the reactionmixture was incubated for 1 h. Solvent was removed by evaporation andthe product was isolated on RP HPLC. Appropriate fractions were pooled,evaporated to dryness, co-evaporated with MeOH and the product wasrepurified on a preparative TLC plate (1×250×250 mm, elution withisopropanol—water—conc. NH₄OH (15:4:1)). The product-containing band wasscratched out and the product was eluted with MeOH—water (7:3) mixture.MS ES⁺ 231.0 [M+H⁺], 253.2 [M+Na⁺], yield 1.7 mg (67%). MS ES⁺ 231[M+H⁺].

Example 4f Synthesis of1-(2-Deoxy-α-D-ribofuranosyl)-4-amino-1,2,5,6-tetrahydro-1,3,5-triazin-2-one(10)

[0338] Compound 10 was synthesized by analogy to the preparation of 9 bydeprotection of 8 with ammonia using method B. Compound 10 was obtainedas a solid.

Example 4g Synthesis of3′,5′-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-5,6-dihydro-N⁴-isobutyryl-5-aza-5-N-methylcytidine(15)

[0339] 18 mg of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-5,6-dihydro-N⁴-isobutyryl-5-azacytidine,0.1 mL of N,N-diisopropyl-N-ethylamine and 1.0 mL of dimethylsulfatewere incubated for 1 h at room temperature. The product was isolated byflash chromatography on silica gel (MeOH gradient in dichloromethane).ES⁺ MS signals at 573.2 [M+H⁺], 595.3 [M+Na⁺] and 1167.2 [2M+Na⁺], yield14 mg (77%).

Example 4h Synthesis of 5,6-Dihydro-5-aza-5-N-methylcytidine (16)

[0340] 14 mg of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-5,6-dihydro-N⁴-isobutyryl-5-aza-5-N-methylcytidinewas dissolved in 2 mL of MeOH and treated with 6 mL of concentratedNH₄OH during 16 h at room temperature. The solution was evaporated todryness, co-evaporated with toluene and dissolved in 2 mL of THF. To thesolution 0.5 mL of 1 M tetrabutylammonium fluoride was added and thereaction mixture was incubated for 1 h. Solvent was removed byevaporation and the product was isolated on RP HPLC. Appropriatefractions were pooled, evaporated to dryness, coevaporated with MeOH andthe product was repurified on a preparative TLC plate (1×250×250 mm,elution with isopropanol(15):water(4):conc. NH₄OH(1)). The bandcontaining product was scratched out and the product was eluted withMeOH(7):water(3) mixture. MS ES⁺ 261.0 [M+H⁺], 520.9 [2M+H⁺], yield 5.5mg (86%). MS/MS of the 261.0 mass ion generated the expected fragmentwith m/z 128.9, corresponding to the5,6-dihydro-5-aza-5-N-methylcytosine base.

Example 4i Synthesis of3′,5′-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-5,6-dihydro-N⁴-isobutyryl-5-aza-5-N-methylcytidine(17)

[0341] A mixture of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-5,6-dihydro-N⁴-isobutyryl-5-azacytidine(14) (11 mg), 0.1 mL of N,N-diisopropyl-N-ethylamine and 1.0 mL ofdimethylsulfate was incubated for 1 h at room temperature. The product17 was isolated by flash chromatography on silica gel (MeOH gradient indichloromethane). ES⁺ MS signals at 557.3 [M+H⁺] and 579.3 [M+Na⁺],yield 9 mg (80%).

Example 4j Synthesis of 2′-Deoxy-5,6-dihydro-5-aza-5-N-methylcytidine(18)

[0342] A solution of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-deoxy-5,6-dihydro-N-4-isobutyryl-5-aza-5-N-methylcytidine(17) (9 mg) in MeOH (2 mL) was treated with concentrated NH₄OH (6 mL)and kept at room temperature for 16 h. The solution was evaporated todryness, coevaporated with toluene, dissolved in THF (2 mL) and treatedwith a solution of 1 M tetrabutylammonium fluoride in THF (0.5 mL). Thereaction mixture was left at room temperature for 1 h. Solvent wasremoved by evaporation and the product was isolated on RP HPLC.Appropriate fractions were pooled, evaporated to dryness, coevaporatedwith MeOH and the product was further purified on a preparative TLCplate (1×250×250 mm, elution with isopropanol—water—conc. NH₄OH(15:4:1)). The product-containing band was scratched out and the productwas eluted with MeOH—water (7:3) mixture. MS ES⁺ 245.0 [M+H⁺], 267.1[M+Na⁺], yield 18 was 3.7 mg (93%). MS/MS of the 245.0 mass iongenerated the expected fragment with m/z 128.9, corresponding to the5,6-dihydro-5-aza-5-N-methylcytosine base.

Example 4k Synthesis of 6-Methyl-5-azacytosine (19) (Scheme 6).

[0343] 8.4 g of dicyandiamide was suspended in a mixture of 16 mL ofAc₂O and 1 mL of AcOH. The reaction mixture was refluxed during 16 h.After cooling the reaction mixture was evaporated to dryness. Theproduct was isolated on preparative RP HPLC (CH₃CN gradient 0-20% over20 min in 0.1 M triethylammonium bicarbonate buffer (pH 7.0)). Retentiontime was 6.7 min, λmax=237 nm, MS ES⁻=125, yield 30%.

Example 4l 6-Methyl-5-azacytidine (20)

[0344] Compound 20 was synthesized according to (Hanna N. B., ZajicekJ., Piskala A. Nucleosides & Nucleotides 16, 1997, p.129-144) with 8%yield starting from 6-methyl-5-azacytosine (19).

Example 4m Synthesis of 6-Methyl-2′-deoxy-5-azacytidine (21)

[0345] 30 mg of 6-methyl-5-azacytosine, 5 mg of (NH₄)₂SO₄ and 5 mL ofhexamethyldisilazane were refluxed overnight at 125° C. (external oilbath temperature). The clear solution was evaporated to solid andco-evaporated with 5 ml of xylene. To the residue 70 mg of the3,5-bistoluoyl-1-chloro-2-deoxyribose was added and the mixture wassuspended in 2 mL of CH₃CN. Incubation with stirring was continued for24 h and then a mixture of 173 mg AcONa and 0.3 mL AcOH, diluted to 1 mLwith water was added. After 1 h the mixture was diluted with 20 ml ofwater and extracted twice with 20 mL of ethyl acetate. The organic layerwas dried over Na₂SO₄, filtered, evaporated and the products wereseparated by silica gel chromatography. Yield of β-anomer 25%, α-anomer(23%).

[0346] The protected nucleoside was treated with 0.02 M NaOMe in MeOHfor 4 h to remove toluoyl protecting groups. The resulting nucleosideswere isolated on PR HPLC.

Example 4n Syntheses of 6-phenyl-5-azacytosine (22) and6-phenyl-5-azacytidine (23)

[0347] (Scheme 7) were carried out according to published procedure(Hanna N. B., Masojidkova M., Fiedler P., Piskala A. Collect. Czech.Chem. Commun. 63, 1998, p.222-230). Yield of the base was 43%,nucleoside—16%.

Example 4o Synthesis of 5-azacytidine and 6-methyl-5-azacytidineprodrugs (Scheme 8)

[0348] 8.3 mg of the nucleoside was suspended in 1 mL of THF and 11 uL(4 eq.) of N-methylimidazole was added. The reaction mixture was cooledto −78° C. and 4-bromophenyl-N-methoxyalaninylphosphorochloridate (15mg) in 0.5 mL of THF was added dropwise during 30 min. After 1 h another10 mg of the phosphorochloridate was added, the mixture was allowed towarm to room temperature and incubated overnight. The mixture wasevaporated and separated by RP HPLC. 5-Azacytidine prodrug was eluted at23 min (0-20% CH₃CN in 23 min), λmax=226 nm, yield approximately 15%.6-Methyl-5-azacytidine prodrug was eluted at 24 min, λ max 225 nm, yieldapproximately 12%.

[0349] The 5-Azacytidine phospholipid prodrugs are synthesized by scheme9. 5-Azacytidine prodrugs, activated by biological reduction aresynthesized by scheme 10.

Example 4p1-(β-D-Ribofuranosyl)-4-amino-1,2,5,6-tetrahydro-1,3,5-triazin-2-one

[0350] (5,6-Dihydro-5-azacytidine) (23) and 6-oxo-5-azacytidine (24)(Scheme 11) were synthesized by (Beisler, J. A., Abbasi, M. M., Kelley,J. A., Driscoll, J. S. J Carbohydrates. Nucleosides. Nucleotides, 4(5),1977, pp 281-299).

Example 4q

[0351] 2′-Deoxy-5,6-dihydro-5-azauridine (26) is synthesized by areduction of 2′-deoxy-5-azauridine (25) by analogy to the reduction ofcompound 3 to 4 (Piskala A., {haeck over (C)}esnekováB., Veselý J. Nucl.Acids Symp. Ser. No 18 (1978) pp 57-60).

Example 4r Synthesis of 2′-deoxy-5,6-dihydro-5-azacytidine Palmitate(27)

[0352] To a solution of (9) (0.26 g, 1.13 mmol) in MeOH (50 mL) wasadded a solution of palmitic acid (0.29 g, 1.13 mmol) in hot MeOH (10mL) and evaporated. The residue was triturated with ether and filteredgiving 0.57 g (quantitative yield) of a colorless product with m.p.123-124°. MS ES+ 231 [M+H⁺].

Example 5 In Vitro Assays Demonstrate that DHAdC is Safe and EffectiveAgainst HIV Infection

[0353] In Vitro Passaging Assays of DHAdC

[0354] Passaging experiments were performed for DHAdC (also reffered toas SN1212), to demonstrate that viral eradication is possible in vitro.The experiment was carried out in quadruplicate in the presence ofSN11212 at a concentration of 100 nM. Levels of p24 fell permanentlybelow the limit of detection (4 ng/ml) by passage 8. No infectious viruswas recovered after passage 12. (Data not shown.)

[0355] DHAdC is a Viral Mutagen.

[0356] Assessment of DHAdC viral mutagenicity was carried out asdescribed above for 5-aza-dC. Mutagenesis of the sense strand of a 0.9kb fragment of reverse transcriptase of HIV NL4-3 was determined after asingle passage in SN1212 (50 μM) and compared to an untreated control.Results are shown in Table 5. TABLE 5 Mutations/ G→A T→C A→T A→C G→T G→CAnalog Nucleotide % A→G C→T T→A C→A T→G C→G SN1212 37/24,828 0.015 17 121 1 3 3 Control 32/28,658 0.011 27 3 0 1 1 0

[0357] The mutation rate induced by 50 μM SN1212 in HIV RT is 1.4-foldhigher than control (0.0015 in DHAdC treated versus 0.0011 in control).The dominant mutations are C⇄T transitions (enhanced 4.6-fold bySN1212), with a minority of transversions (pyrimidine⇄purine). Incontrast, 5-OH-dC demonstrated only a 1.14-fold increase in overallmutation rate over background.

[0358] DHAdC does not Cause Significant Mutagenesis of Cellular DNA.

[0359] SN1212 is a poor substrate for polymerase-α, the cellularpolymerase responsible for most DNA synthesis. (Data not shown.) Anhgprt assay was also performed to test mutagenesis of cellular DNA byDHAdC. The assay was performed on CHO (Chinese Hamster Ovary) cells andmutants were selected for resistance to 6-thioguanine (6-TG). EMS (ethylmethyl sulfonate), a known mutagen, was used as a positive control.SN1212 at a concentration of 1 mM did not increase above background themutation frequency of a cellular gene, hgprt. (Data not shown.) Of note,the EC₅₀ of DHAdC against HIV is in the range of 10 nM, while nosignificant mutation to cellular DNA is noted at 1 mM, a 10,000-folddifference.

[0360] Mitochondrial toxicity is also a safety concern with nucleosideanalogs. SN1212 was also analyzed for mitochondrial toxicity. SN1212does not demonstrate evidence of mitochondrial toxicity by either anincrease in lactate production or inhibition of mitochondrial DNA at thehighest dose tested, 320 μM. (Data not shown.)

[0361] DHAdC is Effective Against Wild-Type HIV Strains and NRTIResistant HIV Strains.

[0362] The effectiveness of DHAdC was tested against wild-type HIVstrains and NRTI resistant HIV strains as described in Example 2. Thefollowing strains were tested: HIV-1 LAI, wild-type; HIV-ILAI-M184V-M184V mutation with resistance to lamivudine (3TC); HIV-1RTMDR1-74V, 41L, 106A and 215Y mutations with resistance to zidovudine,didanosine, nevirapine and other non-nucleoside reverse transcriptaseinhibitors; and HIV-1 RTMC-67N, 70R, 215F and 219Q with resistance tozidovudine. Results are shown in Table 6. TABLE 6 SN1212 AZT 3TC HIVStrain (EC₅₀) μM (EC₅₀) nM (EC₅₀) nM Wild-type 6 10 45 RTMC 6 300 330M184V 6 10 >32,000 RTMDR1 6 60 N.D.

[0363] The EC₅₀'s of SN1212 were the same in wild-type and the threemutant HIV strains, confirming the lack of cross-resistance betweenSN1212 and NRTI. Furthermore, based on HIV passaging experimentsdesigned to favor the emergence of resistant strains performed withSN1212, it appears unlikely that de novo resistance will develop toSN1212.

Example 6 In Vivo Assays Demonstrate that DHAdC, or Prodrugs Thereof,are Safe and Effective Against HIV Infection

[0364] DHAdC is Effective in Treating HIV Infections in a Mouse Model

[0365] SN1212 was administered at up to 100 mg/kg/day subcutaneously inSCID-Hu Thy/Liv mice for 21 days, without any significant toxicity beingdemonstrated. After completion of this toxicology experiment, SN1212 wastested in HIV infected SCID-Hu mice. While SN1212 did not demonstratereduction in p24 or HIV RNA, it demonstrated a significant decrease inviral infectivity when compared to untreated animals at a dose of 10mg/kg (see, e.g., Table 7). The discordance between viral infectivityand conventional surrogate markers of viral load, such as p24 or HIVRNA, is not surprising, as it has also been observed in vitro, andreflects the increased proportion of non-infectious viral particles inthe presence of SN1212. It is also interesting to note that, of thetreated groups, the immunologic profile of the SN1212 groups mostclosely resemble that of the uninfected group. This is compatible withthe finding that infection with less “fit” viruses provides a relativeclinical benefit by preserving cellular immunity. TABLE 7 HIV-1 ViralCD4 + p24 RNA liter Drug/Dose CD8 + CD4+ CD8+ (pg/10⁶ (log copies/(viruses/ (mg/kg/day) (%) (%) (%) cells) 10⁶ cells) 10⁶ cells) SN1212(100) 63 10 11 570 5.1 24.9 SN1212 (10) 70 13 9.5 510 5.3  15.2** 3TC(30) 77 6.9 4.2  0**  2.0**  0** NDC* 63 7.9 5.9 250 5.4 66.5 Uninfected65 12 9.4 0 0 0

[0366] A Prodrug of SN1212, SN1461, is not Toxic in Animals.

[0367] SN1461 is a prodrug that in humans is converted predominantly inthe liver to SN1212. SN1461 has been tested for pharmacokineticcharacteristics in a number of animal species. In rats, SN1461 has ahalf-life of 3.9 hours and an oral bioavailability of 43%, while inbeagles; SN1461 has a half-life of 2.1 hours and an oral bioavailabilityof 51%, prior to formulation enhancements. A single dose of up to 1 g/kgof SN1461 has been given orally to rats and up to 2 g/kg to dogs in adose escalation study without evidence of toxicity.

[0368] The present invention provides a novel class of mutageniccompounds, and methods of using and preparing these compounds. Whilespecific examples have been provided, the above description isillustrative and not restrictive. Any one or more of the features of thepreviously described embodiments can be combined in any manner with oneor more features of any other embodiments in the present invention.Furthermore, many variations of the invention will be apparent to thoseskilled in the art upon review of the specification. The scope of theinvention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

[0369] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not imply that any particular reference is“prior art” to their invention.

What is claimed is:
 1. A compound having the formula:

wherein Y is a member selected from C, CH and N; Z is a member selectedfrom C, CH and B; R¹ is a member selected from H, acyl, OR⁹, SR⁹, NHNH₂,NR⁹R¹⁰, ═O and ═NR⁹, wherein R⁹ and R¹⁰ are members independentlyselected from H, substituted or unsubstituted alkyl, acyl, substitutedor unsubstituted heteroalkyl and substituted or unsubstituted aryl; R²is present or absent and is a member selected from H, acyl, substitutedor unsubstituted alkyl, OR¹¹, SR¹¹, NR^(11a), NR^(12a), halogen, and ═O,wherein R¹¹ is a member selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heterocycloalkyl, and substituted orunsubstituted heteroaryl; R^(11a) and R^(12a) are members independentlyselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl; R³ is a member selected from H,acyl, substituted or unsubstituted alkyl, NR¹²R¹³, NR¹²OR¹³, SR¹², (═O)and OR¹², wherein R¹² and R¹³ are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl; R⁴ and R^(4a) are members independentlyselected from H, halogen, OMe and OH; R⁵ and R⁶ are membersindependently selected from H, and OR⁴, wherein R¹⁴ is a member selectedfrom H, substituted or unsubstituted alkyl, acyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl andP(O)(R¹⁵)(R¹6), wherein R¹⁵ and R¹⁶ are members independently selectedfrom OR¹⁷, NR¹⁷R¹⁸, OCH₂CH₂CN, substituted or unsubstituted alkyl andsubstituted or unsubstituted nucleosides, wherein R¹⁷ and R¹⁸ aremembers independently selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl, whereina member selected from R⁵ and R³; R⁶ and R³; and R¹⁵ and R¹⁶ togetherwith the atoms to which they are attached are optionally joined to forma ring system selected from substituted or unsubstituted cycloalkyl andsubstituted or unsubstituted heterocycloalkyl; R⁷ and R⁸ are eitherpresent or absent and are independently selected from H, acyl,substituted or unsubstituted alkyl, and R¹ and R⁸, together with theatoms to which they are attached are optionally joined into a ringsystem selected from substituted or unsubstituted cycloalkyl andsubstituted or unsubstituted heterocycloalkyl.
 2. The compound accordingto claim 1, having the formula:


3. The compound according to claim 1, having the formula:


4. The compound according to claim 3, wherein R¹¹ is a member selectedfrom silyl groups and substituted or unsubstituted alkyl ethers.
 5. Thecompound according to claim 1, having the formula:


6. The compound according to claim 5, having the formula:

wherein R¹⁹, R²⁰, and R²¹ are members independently selected from H,acyl and substituted or unsubstituted alkyl.
 7. The compound accordingto claim 5, having the formula:


8. The compound according to claim 1, wherein R⁶ has the formula:

in which R²² is a member selected from substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl; L is a linkerselected from substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; and Ar is a member selected from substitutedor unsubstituted aryl and substituted or unsubstituted heteroaryl. 9.The compound according to claim 8, wherein L comprises a moiety that iscleaved in vivo after entry of said compound into a cell.
 10. Thecompound according to claim 1, wherein R⁶ has the formula:

in which R²² is a member selected from substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl; L is a linkerselected from substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; and n is an integer from 1 to
 30. 11. Thecompound according to claim 10, wherein L comprises a moiety that iscleaved in vivo after entry of said compound into a cell.
 12. Aformulation of the compound according to claim 1, and a second compoundhaving the formula: A-B wherein A is a hydrophobic domain; and B is ahydrophilic domain covalently bound to A.
 13. The formulation accordingto claim 12, further comprising a polycationic species.
 14. Theformulation according to claim 13, wherein said polycationic species isa dendrimeric polyamine.
 15. The formulation according to claim 12,wherein said formulation is an aqueous formulation.
 16. A method fortreating a viral disease comprising administering to a subject in needof such treatment a therapeutically effective amount of a compoundaccording to claim
 1. 17. The method of claim 16, wherein said compoundis given orally.
 18. The method of claim 17, wherein said compound is anenteric formulation.
 19. The method of claim 18, wherein said compoundis delivered in an oral osmotic drug delivery device.
 20. The method ofclaim 16, wherein the viral disease is caused by a virus that is amember selected from RNA virus and DNA virus.
 21. The method of claim16, wherein the viral disease is caused by a retrovirus.
 22. The methodof claim 21, wherein the viral disease is caused by HIV.
 23. The methodof claim 22, wherein the HIV is resistant to nucleotide reversetranscriptase inhibitors.
 24. The method of claim 16, wherein the viraldisease is caused by a virus of the Flaviviridae family.
 25. The methodof claim 24, wherein the viral disease is hepatitis C.
 26. The method ofclaim 16, wherein the viral disease is caused by a virus of theParamyxoviridae family.
 27. The method of claim 20, wherein the DNAvirus is hepatitis B virus.
 28. The method of claim 20, wherein the DNAvirus is smallpox/vaccinia virus.